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A new electron gun has been built which features mechanical and optical simplicity. Theoretically, it can produce a focused spot having a radius smaller than 50 Å and provide 1000 times more intensity than a hot filament system having a similar final spot size. The increase in intensity is made possible by using a field emission electron source operating at a pressure of 10−9 Torr, which is provided (without baking) using commercially available pumps. The small spot is produced by using two properly shaped electrodes which accelerate and focus the electrons from the tip. It would take a hot filament gun and at least two additional lenses to replace this field emission gun when a spot radius less than 100 Å is required. Even then the brightness of the conventional source would be too low to make use of the small spot size obtained. The optical properties for the new gun were predicted on a computer and experimentally confirmed in a new scanning electron microscope. The aperture aberration coefficient was measured to be no more than a factor of two greater than the theoretical value of 1.5 cm. A spot radius of 250 Å has been measured, and this value is to be compared with the theoretical value of 150 Å. Although it was convenient to measure the spot directly only at a relatively large image distance (11.3 cm), calculations imply that the gun can provide a spot radius less than 25 Å when very small image distances are used. The gun can be used in pulsed operation because all optical properties are constant for a given voltage ratio so that application of the electrode voltages by means of a voltage divider provides automatic focusing for arbitrary changes in the applied voltage. The methods used to make and operate reliable high field emission tips are reviewed, and a technique is described for changing the required tip voltage to obtain a given emission current.

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The complete dissociation of the hexagonal bilayer structure ofLumbricus 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 m (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 H, HI, and IV) and that peak II consisted of subunit M (16 kDa, chain 1). When dissociation was incomplete, two additional peaks were present, peak Ia eluting at the same volume as the whole hemoglobin and peak lb (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. Peak lb contained all four subunits but was usually deficient in subunits D1 and D2, was not always in equilibrium with the whole molecule, and could be dissociated further into II and HI. The patterns of dissociation observed at neutral pH were very similar to those observed previously at alkaline pH and at acid pH and appear to be incompatible with the generally accepted multimeric model of Lumrbncus hemoglobin subunit structure. A model is proposed in which it is postulated that the stoichiometries of some of the subunits need not be constant and that subunits D1 and D2 either form a "bracelet" decorated with complexes of T and M subunits or serve as "linkers" between the latter, to provide the appearance of a two-tiered hexagonal structure. Additional support for the proposed model comes from observations that the fragment II obtained subsequent to dissociation at pH 4, in sodium phosphotungstate, in sodium perchlorate, and in potassium thiocyanate was found to be in equilibrium with a hexagonal bilayer structure IaR(II), whose dimensions were =20% smaller than those of the native hemoglobin.

Experiments with this scanning microscope have produced extremely encouraging results so that we feel Confident in predicting high resolution and high contrast after some obvious modifications are made in the system, such as providing a good objective lens. Experience with conventional lenses indicates that the instrument behaves in a predictable manner and there is no reason to doubt that the resolution can be as good as that of a conventional microscope. The use of quadrupole lenses will depend on calculations now being performed. There is cause for optimism; high resolution may also be possible with this kind of lens. Experience with field emission shows that the technology is not difficult and that there is more than enough current available for any conceivable use. Energy-loss measurements have been made on a variety of materials. It is attractive to consider the possibility of chemical analysis of selected areas of a specimen. We believe that a very crude form of analysis may indeed be possible. The principal advantage of the use of energy-loss techniques, however, may be in the availability of another contrast mechanism. The ability to "see" small details may be considerably enhanced. Finally, we are experimenting with the possibility of using transmitted electrons of different energy losses to produce different colors on a color television display. This should add an extra element to the picture contrast which may be of some value.

SUMMARY The scanning transmission electron microscope is of quite recent origin, and it is only in the last few years that it has been shown that this instrument is capable of giving the same high resolution as the conventional electron microscope. In this article we examine the conditions necessary for the achievement of high resolution and also the various modes of contrast which can be obtained from this instrument. Finally, we suggest other ways in which the microscope can be used in future investigations.

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. The first peak, which was eluted at a slightly greater volume than the native Hb, consisted of reassociated hexagonal bilayer structures when examined by scanning transmission electron microscopy. The dimensions of the two reassociated hexagonal bilayer structures were a vertex-to-vertex diameter of 25 nm and a height of 16 nm. The difference in size between the hexagonal bilayer structures and the native Hb is the contribution of subunit M, which consists of a single hemecontaining chain I (16.75 kDa). Although the reassociated hexagonal bilayer structures have overall dimensions smaller than the 30 nm x 20 nm dimensions of the native Hb, the diameters of the central cavities are not substantially altered. Subtraction of the three-dimensional reconstructions of the reassociated hexagonal bilayer structures from those of the native Hb showed that subunit M was primarily localized at the periphery of Lumbricus Hb. The formation of hexagonal bilayer structures in the complete absence of subunit M provides additional support for the "bracelet" model of the quaternary structure ofLumbricus Hb proposed recently by us in which subunits D1 and D2 were assumed to act as linkers for complexes of subunits M and T or to form a "bracelet" decorated with 12 complexes of subunits M and T.