As part of our long-range planning for the Boulder 3-D facility, we are attempting to evaluate whether an IVEM can yield tomograms of the quality we have come to expect from our HVEM. We have examined 300 nm sections from a PtK cell in metaphase in two JEOL 4000 microscopes operating at 400 KV, one at the National Center for Microscopy and Imaging Research in San Diego, the other at the Lawrence Berkeley National Laboratory. One section of a serial set was imaged in each location; both were adjacent to a section from which a full dual-axis tomographic tilt series had been obtained on our HVEM. Images of a kinetochore and associated microtubules were taken at 12o tilt intervals up to 60o. Negatives were digitized at the pixel size we commonly use for tomography. Images were analyzed by computing a rotationally averaged Fourier transform and examining the power at half-Nyquist and at Nyquist relative to the power at low spatial frequencies. The falloff in power to either side of the tilt axis in images at high tilt was used to measure depth of field. Micrographs from the San Diego microscope had a depth of field comparable to those from our HVEM, whereas those from the Berkeley microscope had somewhat less. The amount of high frequency power as a function of tilt angle was also used to assess how well the microscopes imaged fine detail for progressively thicker specimens. It became evident that different sections gave different amounts of power, even on our HVEM; thus an absolute comparison of the microscopes was not possible. In general the IVEMs appeared capable of yielding the same image quality as does our HVEM with an effective section thickness of 600 nm (300 nm at 60x tilt). The results from these experiments will guide further studies in several ways. It is important to be aware of the effect of aperture size on depth of field, and to include electron dose to the specimen as a parameter to be controlled. Pictures should not be taken at a low dose level, where electron noise in the image dominates the high-frequency information. It is necessary either to do microscopy on exactly the same sample in different microscopes, or to study a stereotyped sample (e.g., muscle). The evaluations must be done with thicker sections, e.g., 0.5 to 1.0fm. If image-forming capability cannot be adequately characterized as a function of sample thickness, it may be necessary to do full tomographic reconstructions on each microscope to settle these issues.
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