PubMed · 14989646
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Greg Dobbin. 2003. Cutting cryostat sections.. https://doi.org/10.1080/10520290310001624617
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The feasibility of using a focused ion beam (FIB) for the purpose of thinning vitreously frozen biological specimens for transmission electron microscopy (TEM) was explored. A concern was whether heat transfer beyond the direct ion interaction layer might devitrify the ice. To test this possibility, we milled vitreously frozen water on a standard TEM grid with a 30-keV Ga(+) beam, and cryo-transferred the grid to a TEM for examination. Following FIB milling of the vitreous ice from a thickness of approximately 1200 nm to 200-150 nm, changes characteristic of heat-induced devitrification were not observed by TEM, in either images or diffraction patterns. Although numerous technical challenges remain, it is anticipated that 'cryo-FIB thinning' of bulk frozen-hydratred material will be capable of producing specimens for TEM cryo-tomography with much greater efficiency than cryo-ultramicrotomy, and without the specimen distortions and handling difficulties of the latter.
Therapeutic success and prognosis in RRP is determined by negative surgical margins. In order to minimize the proportion of positive surgical margins in the final histological evaluation, valid intraoperative control by means of frozen margin analysis is indispensable. We have developed and evaluated a new frozen margin procedure based on the Stanford method with transverse and sagittal cut directions. This technique facilitates comprehensive intraoperative evaluation of curved margin areas for the first time. Retrospective analysis of the results of the new frozen section technique revealed positive surgical margins in 2.7% of patients. The results obtained with this new technique were significantly superior to those obtained with two established techniques (10.3%, P < or =0.001; 17.2%, P < or =0.001). Our results demonstrate that the new frozen margin technique is clearly more sensitive for intraoperative detection of positive margins and thus leads to substantially higher rates of negative surgical margins.
In immunofluorescence microscopy (IFM), the repression of out of focus fluorescence signal is crucial in order to obtain high-resolution images. One option to acquire high vertical resolution (z-axis resolution) is to produce optical sections with a confocal microscope. The z-axis resolution usually obtained with confocal microscopy of biological samples is about 500 nm. Another option is to produce very thin sections with a cryo-ultramicrotome (physical sections). The ultrathin cryosections we employ are about 100 nm in thickness: thus all of the fluorescence must come from within this 100 nm thickness. The use of ultrathin cryosections permits the acquisition of extremely high-quality images and minimizes the possibility for false localization in IFM (Fig. 1). Ultrathin cryosections can be applied to immunoelectron microscopy (IEM) as well as IFM (Fig. 2). We show new methods of ultrathin cryosection immunocytochemistry(1-3). Human full-term placentas were fixed with 4% paraformaldehyde, solidified with 10% gelatin, infiltrated with 2.3 M sucrose, and then frozen in liquid nitrogen. Ultrathin cryosections were cut with a cryo-ultramicrotome and then transferred to glass cover slips for IFM or to nickel grids for IEM. Cryosections were incubated with mouse anti-p230, a trans-Golgi network marker, and subsequently incubated with Alexa 488-labeled goat anti-mouse IgG or with goat anti-mouse 5-nm colloidal gold particles. For visualization and preservation of ultrastructure of cryosections at the electron microscopic level, the sections on grids were postfixed with ferrocyanide-reduced osmium and then stained with uranyl acetate and lead citrate in polyvinyl alcohol(1). Ultrathin cryosection immunocytochemistry should be an important technique for functional genomics research, especially for the analysis of the in situ expression of target molecules(2,3).