Specimen Drift Research Articles

Beam induced mass loss in high resolution biological microanalysis.

Electron beam induced loss of mass from the organic matrix and from higher Z constituents of biological samples was measured by monitoring bremsstrahlung and peak changes in EDS spectra. When any effects of contamination, extraneous X-rays, beam current drift, specimen drift, and specimen shrinkage were monitored and corrected for, the three types of samples gave consistent and similar results at 296 K. Bremsstrahlung losses averaged 45%, 46% and 50% respectively for muscle homogenate, salivary gland sections and albumin. Sulphur losses average 74%, 72% and 86% for the same three sample types. No other elements suffered significant losses. D1/e for bremsstrahlung averaged 0.14 C/cm2. Bremsstrahlung loss at 93 K began approximately one order of magnitude higher in dose, and the extent of loss varied. Sulphur losses, however, were greatly reduced at low temperatures.

Experimental Determination of the Imaging Properties of a 200 kV Electron Microscope

Optical transforms from a through focal series of images of amorphous films of Ge were used to measure the spatial frequencies of maximum and minimum phase contrast of a specific 200 kV lEOL electron microscope. This information was used to determine precise values for the spherical aberration coefficient and defect of focus. Under the appropriate conditions of lens excitation the spherical aberration coefficient was found to be as low as 0�94 mm. Other image defects revealed with great precision were associated with astigmatism, beam divergence and specimen drift in the microscope stage. Quantitative examples illustrating these effects are discussed.

On the detection and size classification of nanometer-size metal particles on amorphous substrates

The detection and size analysis of small metal aggregates supported on amorphous substrates becomes increasingly difficult when the particle size approaches that of the phase contrast background structures of the support. Standard conventional transmission electron microscopy (CTEM), even with subsequent analog image analysis, becomes inconclusive or fails for particles less than 1 nm in diameter, and clear differentiation of the particles from the background can only be made with considerable effort, typically involving several micrographs taken from the same specimen area under different imaging conditions. The TEM image contrast transfer mechanism is briefly reviewed with emphasis on practical conclusions. In the 1–2 nm particle size range, particles can be distinguished from the amorphous background only when focus, astigmatism, and specimen drift are optimally controlled. Operating at 100 keV and with C s = 2.2 mm, a deviation of 100 nm from the optimum focus condition was, for example, found to increase the “apparent” diameter of 1.3 nm particles by as much as 0.4 nm. Furthermore, the electron exposure required to perform such microscopy may severely complicate the situation by radiation- induced changes in specimen and support. These changes may be intensified by specimen contaminants and an improperly controlled environment. Compared to standard high resolution imaging practice, operation at lower magnifications, lower acceleration voltage, larger illumination aperture, and closer to the Gaussian focus is recommended for routine imaging of very small particles on amorphous substrates.

Some data of a superconducting lens with iron circuit

In our laboratory the electron optical data of several iron free lens types were calculated from their axial field distribution /1/. One of these lens types, the shielding lens, which was especially suitable for our purposes has been constructed for practical application and it is now in operation in several electron microscopes. A point to point resolution of 0.15 nm could be established and the specimen drift could be reduced in comparison with conventional microscopes to 0.03 nm/min. The shielding lens is particularly of advantage for high voltage microscopes. But the performance is also satisfactory in the 100 kV range.The shielding material, however, we use for our shielding lens is not commercially available and the machining of this material is difficult. For this reason we wanted to find out if in principle a superconducting iron circuit lens could replace the shielding lens in our system (Fig. 1) at lower beam voltages, i.e. if an iron circuit lens with the same dimensions and the same electron optical data could be constructed.

Intelligent interface for a microprocessor controlled scanning transmission electron microscope with x‐ray imaging

AbstractAn intelligent interface has been designed to perform synchronous digital image acquistion and scan generation (SDIASG interface) for a microprocessor controlled Scanning Transmission Electron Microscope (S(T)EM) with x‐ray imaging. The SDIASG interface connects an LSI‐11/2 microprocessor to a Philips EM400 electron microscope. The LSI‐11/2 microprocessor is part of a DeAnza VC5000 digital image display system. A system using the SDIASG interface is described. The system takes advantage of the SDIASG interface and a DeAnza VC5000 digital image display system to realize new capabilities that optimize conditions for x‐ray mapping.A low characteristic x‐ray count rate is generated by the ultrathin specimens from which high resolution x‐ray maps can be obtained (Shuman et al, 1976; Somlyo and Shuman, 1982). This low count rate necessitates a long image accumulation time, which in turn makes drift correction essential for maintaining spatial resolution. The new capabilities of the system described here consist of real‐time display and summation of consecutive image and x‐ray maps, and automatic return to a high speed imaging mode between consecutive x‐ray map passes. The new capabilities combine to allow frequent correction for specimen drift between consecutive x‐ray mapping passes while still permitting a long total accumulation time for the x‐ray maps.

Ultrastructural imaging of whole cells by cryo-HVEM

A cryostage cooled by liquid nitrogen has been recently completed and activated on the top-entry 1 MV JE0L JEM-1000 of the National Facility for high-voltage electron microscopy in Boulder, Colorado. It provides continuous and controlled cooling of a specimen during observation at an arbitrary temperature in the range 78K to room temperature. Good thermal stability of about ± 0.1K can be achieved by maintaining an appropriate balance between refrigerant flow and heat provided by an automatically controlled built-in heater. Consequently the overall specimen drift is reduced so that high-resolution lattice images of graphitized carbon (spacing 0.34 nm) and Sn/SnTe alloy (spacing 0.31 nm) can be obtained throughout the temperature range.The interest of such cryoimaging is primarily to view hydrated specimens in frozen form, to explore the advantages of fast freezing and freeze-drying as an alternative to chemical fixation and dehydration, and possibly to benefit from a certain reduction in radiation damage known to take place at low temperatures. Frozen specimens are placed onto the cold cryostage in about 5 minutes with a mechanism developed specifically for this purpose. Thus the specimens are kept continuously immersed in liquid nitrogen, and unexposed to the atmosphere, between freezing and insertion into the air lock leading into the instrument column.

Microanalysis with high Spatial Resolution

ABSTRACTElemental microanalysis, using x-rays and electron energy loss scattering, has been shown to be possible with electron beam probe sizes down to 0.5nm. This paper will discuss some practical problems, such as specimen drift, signal magnitude, and probe-specimen interaction when the probe is made very small. These problems have arisen in two studies: 1) an investigation of as segregation in poly-crystalline Si and 2) imaging of metal spheres with surface and bulk plasmon inelastic scattering.

Improved low dose technique for high resolution electron microscopy

SUMMARYThe use of low‐dose electron micrography can result in unacceptable focusing errors if the specimen plane between focus and micrography areas is tilted. The corrective method applied here involves the use of two focus areas symmetrically disposed with respect to the area to be recorded. A reasonable estimate of correct focus can be made by interpolation between the two known settings.Annular illumination applied to the specimen immediately prior to recording is recommended to reduce any likelihood of specimen drift caused by changing thermal gradients.The methods involve only minor modifications to existing circuitry, are easy to implement, and have resulted in a dramatically improved percentage of well‐focused micrographs.

Computer Aid for the Scanning Transmission Electron Microscope

Analytical Electron Microscopy has been made possible through large technological advances in electron microscopes and in their associated data taking equipment. Among these advances are successful field emission electron sources, allowing reasonable current densities in small probe sizes; and computer based data acquisition systems for characteristic x-ray (EDX) and electron energy loss (EELS) detection, allowing simple viewing and easy processing for elemental analysis. These advances tend to bring new and complex problems of their own, however. For instance, a field emission source routinely produces a 3.5 to 5 Å sized probe in the VG Microscopes, Ltd. HB5 STEM. But a specimen drift of about 20 Å per minute, coupled with a 60 Hertz beam deflection of about 10 Å in an ambient magnetic field of about 4 mG, makes it difficult to confidently analyze areas smaller than about 50 Å in diameter during a ten minute experiment. Problems with presently available data acquisition systems arise from the need to synchronize the data taking device with the microscope.

Improving the image of delicate and complex biological surfaces

AbstractAn initial thermal evaporative coating with Au/Pd protects delicate biological surfaces from thermal damage caused by diode sputtering, allowing additional coating if necessary by the quicker and more complete sputtering method. Additional coating by either method (1) reduces charging and bright areas, (2) improves the physical stability of entire samples and of delicate cell features, and (3) may provide sharper images at high magnification by reducing subtle specimen drift. Most consistent elimination of charging occurred when 10 nm or more of Au/Pd was sputtered on specimens showing charging when previously coated with 30 nm of Au/Pd by thermal evaporation. Aquatic microcommunities, particularly those laden with silt, were characterized by extensive interstitial space so that adequate electrical conductivity required several additional sputter coatings.

High Resolution Side-Entry Goniometer

An analytical electron microscope equipped with a side-entry goniometer (SEG) has recently become more widespread than a conventional electron microscope by the following reasons: (1) a variety of specimen holders, (2) large tilting angle with eucentricity. However, the resolution of SEG-system is about 0.4 nm, whereas the resolution of 0.25 nm or less can be obtained by an electron microscope equipped with a top-entry goniometer (TEG)1). Factors determining the resolution of an electron microscope are (1) the aberration coefficients of the objective lens, (2) stability of exciting currents, (3) illumination angle of the electron beam on the specimen, (4) energy spread of the electron beam, and ( 5) vibration and specimen drift. It has been usually difficult to observe high resolution images during use of the SEG system, because of the aberration coefficients of the objective lens, vibration and specimen drift. In order to obtain a resolution of less than 0.3 nm with SEG system at 200 kV, both of spherical and chromatic aberration coefficients should be reduced less than 2 mm. Moreover, relative amplitude of vibration between the specimen and pole pieces should be less than a half value of resolution limit. The image drift should be less than 0.02 nm/sec, because the exposure time usually required for photographing a high resolution image is about 5 second.

Reduction of radiation damage in an electron microscope with a superconducting lens system.

OBSERVATION of biological specimens at high resolution by electron microscopy is limited by radiation damage. In general, structural details of non-periodical arrangements can only be identified for doses of radiation up to 10−3 C cm−2, corresponding to 63 e nm−2, which restricts the resolution to approximately 2 nm (refs 1,2). Many methods have been applied in an attempt to reduce radiation damage, including high voltage, high vacuum and specimen pretreatment. The most successful, however, was the reduction of the radiation dose, used for certain specimens with periodical structure, where the information could be obtained by computer-aided image enhancement from underexposed films3. In special cases, although it was possible to obtain information at high resolution, the specimen was partly destroyed4. The advantages of low temperatures were also investigated, but due to the lack of an adequate instrument, no agreement between the results could be obtained. Furthermore, little attention was paid to the significance of the dose rate at low temperatures. We now have in operation an electron microscope with a superconducting lens system in which the specimen is cooled to liquid helium temperature5. The most important features of this instrument are the high resolution, which allows the transfer of space frequencies corresponding to the inverse of 0.15 nm, the mechanical and electromagnetic stability (specimen drift 0.01 nm min−1) and the ultra high vacuum around the specimen. These features are necessary for testing the effect of radiation damage. We report here experiments which demonstrate that radiation damage is considerably reduced at low temperatures in a dose rate-dependent manner.

An improved energy analysing electron microscope

An electron spectrometer has been attached to a Siemens Elmiskop I electron microscope in order to combine the facilities of microanalysis and conventional high magnification transmission electron microscopy of metals and alloys. Modifications have been made to the original design described by Cundy, Metherell and Whelan to improve both mechanical stability and microscope vacuum. The resolution of the equipment is 12 AA (Fresnel fringe), the specimen drift rate is 0.5 AA s-1 and the contamination rate of the specimen is less than 5 AA min-1. For typical exposure times the spatial resolution of the equipment is limited to about 100-200 AA by specimen drift.

Ultracryotomy: a technique for cutting ultrathin sections of unfixed frozen biological tissues for electron microscopy.

SUMMARYAn apparatus is described which facilitates the sectioning of unfixed and unembedded biological tissues for electron microscopy. The specimen is maintained at the temperature of boiling liquid nitrogen; the knife temperature may be controlled between −50 and − 150°C. Under the worst operating conditions specimen drift with respect to the knife is about 5 nm/sec, and suggestions are made on how this drift may be further reduced. We propose to call the apparatus an ‘ultracryotome’.Electron micrographs of a variety of unfixed, unembedded and unstained tissues are shown and commented on. Some developments of experimental electron microscopy which may be expected to develop from use of the ultracryotome are listed.

A New High Resolution Transmission Electron Microscope with Second-Zone Objective Lens

A New High Excitation Objective Lens (Second-Zone Objective Lens) was discussed at Twenty-Sixth Annual EMSA Meeting. A new commercially available Transmission Electron Microscope incorporating this new lens has been completed.Major advantages of the new instrument allow an extremely small beam to be produced on the specimen plane which minimizes specimen beam damages, reduces contamination and drift.

Electron Microscopic Observation of Crystal Lattices on the Level with Atomic Dimension

Many electron micrographs showing various lattice images were obtained from single crystal gold films. Many of them showed more than two sets of lattice images side by side or/and crossing to each other, e.g. (200), (020) and (220) crossing to each other. To obtain such images the crystal must be oriented so that Bragg reflections are simultaneously excited for all the lattice planes to be imaged. A new tilted illumination method developed by the present author was used to minimize the chromatic aberration for each of the lattice images. The astigmatism and specimen drift were eliminated along any directions, although for images along only one direction those in the same direction may be allowed. Double films of gold-gold and gold-copper were also studied. They showed moiré fringes besides lattice images.