This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Electron microscopic study of frozen-hydrated biological material avoids the necessity for chemical fixation, dehydration, and staining, and thus provides a view of the specimen in a ?near-native? state. The water in biological specimens must be frozen in ?vitreous? or amorphous form in order to avoid nanometer-scale damage to the specimen due to ice crystal formation. For tissue, the preferred method is high-pressure freezing, due to the depth of good freezing that can be obtained. The frozen tissue must be maintained below the de-vitrification temperature (~-140?C) throughout ultramicrotomy and microscopy. Steady progress has been made over the past two decades in cutting frozen-hydrated sections. Although it remains a challenging task, improved cryo-ultramicrotomes and diamond knives, together with the collective experience of the few laboratories engaged in this work, provided a good starting point for investigators wishing to make use of frozen-hydrated sections. In April, 2002, we were the first laboratory to obtain electron tomograms of frozen-hydrated sections (from high-pressure frozen rat liver tissue). The most important finding was that the interior of the section was free of surface artifacts, thus good 3-D information could be obtained. ? Hsieh, C.-E., Marko, M., Frank, J., and Mannella, C.A. 2002. Electron tomographic analysis of frozen-hydrated tissue sections. J. Struct. Biol. 138:63-73. We did subsequent work in three areas: improvements in high-pressure freezing, comparison of frozen-hydrated and freeze-substituted material, and improvements in section attachment to grids. Rat liver tissue was frozen using needle biopsy kits, with which it was possible to freeze tissue within 40 sec of blood flow cessation. Some tissue was freeze-substituted and embedded in plastic. The main differences between the two techniques related to the relative contrast of cellular components. Tomograms of frozen-hydrated sections that showed excellent structural preservation and also exhibited good sectioning quality, with few surface artifacts (crevasses). This correlation prompted us to compare electron diffraction patterns of earlier tomograms with those the new ones, to test the hypotheses that the improved cutting quality was due to a lower content of microcrystalline ice after high-pressure freezing. However, signs of crystalline ice were not observed in either case. We investigated means to reduce the irreversible compression that occurs in the sectioning direction. Based on recommendations in the literature, we started tested both an oscillating 35 cryo diamond knife and a 25 diamond knife, but no improvement in compression was seen in initial tests. One of the major problems with tomography of frozen-hydrated sections is poor attachment of the sections to the gird. This is due in large part to lack of section flatness, which we documented by low-magnification stereo pairs. We found that sections were attached to Quantifoil grids as well as they were to folding grids, although the grids had to be examined shortly after the sections were cut since sections were easily lost in long-term storage. The thinner Quantifoil grids are advantageous for tomography because they allow more open area at high tilt. Use of the microtome?s glass section press tool, and not the polished metal rod, was required to safely flatten the sections on the Quantifoil grids. We also found that the use of molybdenum grids, reduced wrinkling of the carbon film and may aid in section attachment. In collaboration with Dr. Toh-Ming Lu of RPI, we have started investigating functionalized coatings for TEM grid that may help frozen-hydrated sections to attach more firmly to the grid. In collaboration with Jay McMahon of RPI, we plan to design and fabricate special two-part grids that firmly clamp, and hopefully flatten, the sections.
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