This sub-project involves not only maintaining the microscope, but also investigating how it can be best used to serve the imaging needs of the BMIRR. The IVEM has again performed very well this year with no unplanned downtime. Maintenance items included replacement of the column ion pump, the (accessory) column ion gauge controller, an IC in the beam current measuring circuit, and the oil in the two rotary pumps. A slight leak in the pneumatic valving system was repaired, and the air compressor was serviced. The tilt-rotation stage was repaired by Gatan. The LaB6 cathode was replaced. We have tried two cathodes from FEI, and we are not satisfied with their performance. On the advice of other JEM4000 users, we will change to Kimball Physics cathodes, sold by Barry Scientific. Barry offers cleaning of the Wehnelt and mounting/aligning of the cathode. This avoids the risk of damaging the fragile LaB6 crystal while aligning it, and the difficult task of cleaning the LaB6 deposits off the Wehnelt. A spare Wehnelt assembly is required, however. We should have a spare Wehnelt in any case, since it reduces the time the gun must be exposed to atmosphere during a cathode change, which could result in a time savings of up to two days from shorter pumping and conditioning. The major problem with the microscope is the goniometer. Although, with careful alignment, it is possible to minimize the x-y movement of the specimen to about 1 (m over the whole tilt range, there is a large z-movement, amounting to a total focus change of 10-30 (m. The motorized z-control on the goniometer is not of sufficient mechanical quality be used in the automation system, so the objective lens current must be varied. As mentioned in the above TRD """"""""Development of automated tomography for the IVEM"""""""", this focus change causes several problems with automated tomography. We have confirmed that our focus change is greater than that found in most other labs doing tomography, even those labs using the same model of microscope and goniometer as ours. JEOL has rebuilt the goniometer, and also temporarily exchanged it for a new one, both to no effect. We have been in communication with JEOL in Japan, and they have devised a series of tests to be made by two mechanical experts stati oned in the US. To ensure that bent specimen holders are not causing the goniometer problem, we met with Tom Schmeltzer of Gatan, who detailed a method to determine the straightness of the specimen holders, gave us the tolerance specifications, and told us how to straighten bent holders. Our holders don't seem to be bent at present. A special method of column alignment was devised to prevent shift of the illuminated area when the objective focus changes by a large amount. This eliminated the need of one of the corrections in the automated tomography software. A foot-pedal control was added to stage automation system to make it easier to manually rotate or tilt the specimen and center it at the same time. The goniometer height adjustment range was reset to accommodate both the tilt/rotation holder and the cryo holder. The cryo holder was tested at -180oC at zero and 60o tilt, at both 200 and 400kV. The 3.4A graphitized carbon lines could be seen in all cases. Tests of the imaging performance of the microscope with sections between 0.25 (m and 1 (m in thickness, at zero and 60o tilt, were made. Various TEM and STEM modes were used, and a comparison between CCD and film recording was made. In the TEM mode, it was found that two effects can be minimized by using a smaller objective aperture: blurring of images of very thick sections, and blurring of the edges of high-tilt images. This makes sense according to imaging theory, but the use of apertures even only as small as 10 or 20 (m is difficult. Vignetting of the image area by the aperture at low magnification occurs, and the range of electronic image shift possible before occlusion of the image by the aperture is severely limited. For automated tomography, the full range of electronic image shift must be used to keep the object of interest centered over the whole tilt range. It was found that use of the condenser minilens (CM) can overcome these problems. The optimal CM settings for all accelerating voltages, for either automated tomography or wide-field viewing with small objective apertures were determined experimentally. In the STEM mode, blurring of the edges of high-tilt images could be minimized by using a small condenser aperture to reduce the beam convergence angle. The STEM images were in all cases much better than the TEM CCD images, but about the same as TEM film images. STEM also holds promise for better performance on even thicker sections than those tested, and also for low-dose imaging. The STEM images can be digitized to give 12-bit images 2048x2048 pixels in size with the addition of an A/D converter board in the Tietz system main computer. Sections sent to Dr. M. Kersker of JEOL for imaging with a Gatan Energy Filter-equipped JEM-4000. We are interested in investigating the use of energy filtering to increase in contrast of thick plastic or ice-embedded sections which have no heavy metal contrasting added. Unfortunately, there was not enough time to thoroughly investigate the optimal choice of energy window to use with such specimens. However, we have established a collaboration with Dr. T.S. Kaun of SUNY Albany Physics Dept., and can do more testing there.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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