A flexible instrument control and image acquisition system for a scanning electron microscope.

We have developed an instrument control and image acquisition system for use with scanning electron microscopes. By making the system flexible over a wide range of operating voltages, scan generation and image acquisition modes can be easily accommodated to a wide range of instruments. We show the implementation of this system for use with a custom-built low-voltage scanning electron microscope.

Personal recollection. Some Chicago aberrations.

This article presents my reminiscences of the work in Chicago on the correction or reduction of lens aberrations. Studies began in the early 1960s and extended over a period of almost 40 years, although it was never the primary focus of the work of the laboratory. The account is almost entirely based on my own memory, which is not a very reliable instrument.

First tests of a dipole lens for a scanning electron microscope.

We have previously shown that a dipole lens has superior properties that are particularly suited for use in a low voltage scanning electron microscope (SEM) (Tsai & Crewe, 1996). The aberrations are lower than for any other type of lens and lead to a prediction of high resolution. We describe the construction details of a microscope based on this principle and present some early results.

Studies of a magnetically focused electrostatic mirror. I. Experimental test of the first order properties.

When a uniform magnetic field is superimposed on a uniform electrostatic field, the combination can act as a magnetically focused mirror. This mirror is predicted to have aberrations of opposite sign to those of a magnetic lens and may therefore be useful as a corrector. We have built an electron optical system to test these ideas.

Scanning transmission electron microscopic examination of the hexagonal bilayer structures formed by the reassociation of three of the four subunits of the extracellular hemoglobin of Lumbricus terrestris.

A fraction obtained by gel filtration at neutral pH of the extracellular Hb of Lumbricus terrestris dissociated either at pH 9.8 or at pH 4.0, consisting of the three subunits D1 (31 kDa), D2 (37 kDa), and T (50 kDa), was found to produce two peaks when subjected to gel filtration on Superose 6 at pH 7.

Bracelet protein: a quaternary structure proposed for the giant extracellular hemoglobin of Lumbricus terrestris.

The complete dissociation of the hexagonal bilayer structure of Lumbricus terrestris hemoglobin (3900 kDa) at neutral pH, in the presence of urea, guanidine hydrochloride, sodium perchlorate, potassium thiocyanate, sodium phosphotungstate, and sodium phosphomolybdate, followed by gel filtration at neutral pH on Sephacryl S-200 or Superose 6, produced two fragments, II (65 kDa) and III (17 kDa); NaDodSO4/polyacrylamide gel electrophoresis showed that peak II consisted of subunits D1 (31 kDa, chain V), D2 (37 kDa, chain VI), and T (50 kDa, disulfide-bonded trimer of chains II, III, and IV) and that peak II consisted of subunit M (16 kDa, chain I). When dissociation was incomplete, two additional peaks were present, peak Ia eluting at the same volume as the whole hemoglobin and peak Ib (200 kDa). Scanning transmission electron micrographs of peak Ia showed it to consist of whole molecules and of incomplete hexagonal bilayer structures, missing an apparent 1/12th.

The molecular size of Myxicola infundibulum chlorocruorin and its subunits.

The molecular shape and size of the extracellular chlorocruorin of Myxicola infundibulum was determined using scanning transmission electron microscopy and its dissociation in the presence of sodium dodecyl sulfate (SDS) was investigated using polyacrylamide gel electrophoresis. The shape of the chlorocruorin is that of a two-tiered hexagon with a vertex-to-vertex diameter of 29.0-29.

An introduction to the STEM.

It is particularly appropriate for this Journal to devote one of its issues to the subject of the scanning transmission electron microscope (STEM) and its uses because the initial stimulus for its invention was precisely the problem of discerning ultrastructural details in biological material. To a large extent the capabilities of the instrument have been shown to offer the potential of solving many such problems and what is most needed today is to put such instruments in the hands of biologists together with the information on how and when to use them. This issue should go a long way in satisfying this latter need.

The reassociation of Lumbricus terrestris hemoglobin dissociated at alkaline pH.

The reassociation of the extracellular hemoglobin of Lumbricus terrestris (Mr approximately 3.9 X 10(6) ) at neutral pH, subsequent to its dissociation at pH above 8.0, was examined using gel filtration, ultracentrifugation, and scanning transmission electron microscopy.

Inexact three-dimensional reconstruction of a biological macromolecule from a restricted number of projections.

A computer-generated three-dimensional model of Lumbricus terrestris hemoglobin has been obtained by reconstruction from four STEM low-dose computer-averaged micrographs. X-ray projections of a wooden model constructed from the calculated two-dimensional sections through the molecule were generally consistent with the original STEM projections of the hemoglobin and suggest the presence of large gaps between the central regions of adjacent 1/12th subunits of opposite tiers.

Evidence for a central substructure in a Lumbricus terrestris hemoglobin obtained with STEM low-dose and digital processing techniques.

Applications of scanning transmission electron microscope (STEM) low-dose and digital image processing techniques to the annelid extracellular hemoglobin of Lumbricus terrestris have shown the existence of a central substructure. The techniques involve low-dose STEM images of less than 2 e/A2 irradiation. Many such low-dose images were then aligned in their symmetry, N-fold averaged, and summed.

A low-cost image analysis system for scanning transmission electron microscopy low-dose images.

An image processing system is described which has been used to analyze STEM images of biological macromolecules. The system has the inherent virtues of being inexpensive, easy to program and self-contained. The operating procedures are described using a particular molecule (earthworm hemoglobin) as an example.

Optimal imaging techniques in the scanning transmission electron microscope: applications to biological macromolecules.

We show applications of the optimal imaging method to stained biological macromolecules. This optimal imaging method involves the following basic procedures: (i) for any given resolution, which is represented by the electron probe size in the scanning transmission electron microscope, a preferred magnification is used; (ii) the micrographs taken at the condition described above are then spatially filtered by using a low-pass filter (nu < 1/2d, in which d is the space between scan lines) to optically reconstruct the final optimal image. It is found that the micrographs obtained by using the optimal imaging method clearly show an improvement in contrast.

Dark field imaging of biological macromolecules with the scanning transmission electron microscope.

A scanning transmission electron microscope (STEM) equipped with a field emission gun has been employed for the examination of biological macromolecules at high resolution. The quality of micrographs obtained with the STEM is dependent upon the quality of the substrate used to support biological objects because the image contrast in dark field is proportional to the mass density of the specimen. In order to reduce deleterious effects of the substrates on the image quality, we have developed a method of fabricating substrates consisting of very thin, very clean carbon films supported on very clean fenestrated plastic films.

Direct observations of atomic diffusion by scanning transmission electron microscopy.

The feasibility of using a high-resolution scanning transmission electron microscope to study the diffusion of heavy atoms on thin film substrates of low atomic number has been investigated. We have shown that it is possible to visualize the diffusion of individual uranium atoms adsorbed to thin carbon film substrates and that the observed motion of the atoms does not appear to be induced by the incident electron beam.

Very low voltage electron microscopy.

We conclude that a 150 V scanning microscope with a resolution of 10 A is quite feasible and could have considerable value. It might consist of a field emission source, an electron gun to decelerate the electrons, a condenser lens to produce a parallel beam, a multipole corrector and a short focal length objective lens. Electrons reflected from the specimen surface would pass through a spectrometer whose principal features would be a large collecting power and low (1/200) energy resolution.

The use of backscattered electrons for imaging purposes in a scanning electron microscope.

It is shown that the use of a very large detector for backscattered electrons can provide a signal comparable to that obtained in the secondary emission mode and that the resolution available is also comparable. A technique is described whereby the difference is taken between these two signals, and the use of this difference signal can significantly enhance surface details. It is believed that the use of such a signal should ultimately prove advantageous in increasing resolution.

High resolution imaging properties of the STEM.

The effect of the finite size of the atom on the resolution of the STEM is investigated. When the probe size becomes comparable to the size of the atom, the quality of the image depends on the scattering properties of the atom as well as the distribution of electrons in the probe. A technique for calculating the image of a single atom is developed by expanding the scattering amplitude.