Thick section preparation

Photographs of the various preparations were taken by means of a Philips EM 300 electron microscope. To this end, the samples were embedded in polymethylmethacrylate and 200-400 A thick sections cut from them with an ultramicrotome. 

III. Transmission electron microscopy of radish seeds Transmission electron microscopy (TEM) of radish seeds was done as listed below For TEM preparations, the specimens after fixation and dehydration, were embedded in Epon 812 resin (Luft, 1961). Thick sections (ca. 1mm each) were stained with 0.1% toluidine blue and observed with a Zeiss light photomicroscope. Thin sections, obtained with a diamond knife on a Supernova microtome, were sequentially stained at room temperature with 2% uranyle acetate (aqueous) for 5 min and by lead citrate for 10 min (Reynolds, 1963). Ultrastructural studies were made using a Philips CM12 transmission electrone microscope (TEM) operated at 80 KV. 

Preparation of Thick Sections from EM-Embedded Blocks for Concomitant Light Microscope Radioautography... 

Prepare 1 -pm thick sections by ultramicrotomy (thin sections from ultramicrotomy of same blocks used for obtaining thick sections can be employed for EM radioautography). 

Microscopic analysis. From each experimental group of specimens with incipient lesions, two specimens were selected randomly for microscopic analysis. Two adjacent slices (500 pm thickness) were prepared from each specimen with a diamond wire sectioning machine (model 3242, Well, Le Lode, Switzerland). The slices were fixed in 1.0 ml 4% paraformaldehyde, 1% glutardialdehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for one week. One slice of each pair was subsequently demineralized in 2.0 ml 0.1 M acetic acid, 2.5% glutardialdehyde, pH 4.0. 

Prepare duplicate or triplicate 2- to 3-mm-thick sections of the sample on a microscope slide. For best results, allow sample to solidify directly on the slide so that the surface is sufficiently smooth for imaging. If starting with solidified products, prepare section with a razor blade or a microtome, if possible, for optimal smoothness. 

A scheme of the technical procedure that has proven useful for the concerning studies is shown in Fig. 4 (Pflug, 1984 a)17. Compact and unweathered rock samples are selected for the analyses. The rock sample is sectioned into two halves (top of Fig. 4). From one side of the main cut (lb) a normal ca. 30 pm thin section is produced. From the opposite surface of the cut (la), a thick section is prepared for the purpose of demineralization. The thin section (lb) is used for polarization microscopy and microprobe analyses. 

The sample preparation procedures for the direct analysis of small molecules in tissue have been described by several papers [120-124], Tissues (brain, heart, lung, kidney, liver, etc.), were immediately frozen and stored at -80 °C after harvest. The frozen tissues were subsequently cut into serial 10-20 pm thick section which was typically prepared by cryosectioning on a microtome at a temperature of -20 °C. The adjacent sections were gently mounted onto a conductive surface, MALDI imaging target plate or glass slides. These plates were desiccated under low vacuum for a short period of time until dry, then robotically or manually coated with the... 

The metallic layers were examined either by conventional or cross-section TEM in a Jeol 200 Cx microscope. For the cross section preparation a sandwich of two laminates is made, glued face to face with an epoxy, cut in small pieces, mechanically polished, and then ion milled to a final TEM observation thickness. The plane section TEM sample are prepared by dissolving the PET in trifluoroacetic acid for 5 to 10 mn. The area observed, on plane section TEM, for the grain size calculation is close to 0.2 urn. For the adhesion measurements, test pieces consist of aluminum support (1 mm thick) double sided tape (Permacel P-94) PET (12pm) / evaporated aluminum/ ethylene acrylic acid (EAA) copolymer film. These laminates are prepared for the peel test by compression under 1.3 105 N.m2 at 120°C for 10 seconds. The peel test is performed by peeling the EAA copolymer sheet from the laminate in an INSTRON tensile tester at 180° peel angle and 5 cm min peel rate. 

The green hairy roots cultured in the light for 3 weeks were used for the fluorescent microscopy and transmission electron microscopy (TEM). For the fluorescent microscopy, frozen sections (15 pm in thickness) were prepared using microtome cryostat (HM 500 OM, Microme, Heidelberg, Germany) and sealed with 50% glycerol on glass slides. Localization of chloroplasts was observed by fluorescent microscopy (VANOX AH-3, Olympus). 

Fig. 4.25. TEM image of a 100-nm thick sliver of a TEM window cross-section prepared by microtome slicing supported on a glassy carbon grid (on the right side of the image). The thicknesses of the Si3N4 (30 nm), Si02 (7 nm), and the interfacial layer between the Si and Si3N4 are shown (from [125])...

Ethylene oxide (ETO) Is the predominantly used sterilant In the United States for membrane devices destined for medical use. Conditions for Its use have been well established to assure sterility (51), although some problems arise from the slow diffusion of the gas from thick sections of thermoplastics, such as the headers In hollow fiber devices. The majority of hemodlalyzers are prepared with cellulosic membranes, which are partially... 

In the method developed by Pickel et al. (1976), organs or animals are perfused with fixative, followed by the preparation of sections (about 20 pm) which are then treated with detergent (Triton X-100) to increase permeability. Subseqent EIH staining, as in light microscopy (Section 17.3.3), gives suitable results for the outer 2-3 pm layer of thick sections. 

To facilitate the measurement of the bulk conductivity, the toner powders were compressed into thin cylindrical disks under a hydrostatic pressure of 100,000 psi. Under these pressures the toner flowed readily and formed a uniform pellet in which the toner particle surfaces were in more intimate contact than in a loose powder. Gold electrodes were evaporated onto the flat faces and the current as a function of the applied electric field measured. This measurement method of the conductivity has certain inherent disadvantages, however, this measurement was intended to be used simply to contrast the kinetic conductivity values reported in Section IV, not to extract absolute values of the material parameters. Nonetheless, several samples of various thicknesses were prepared and measured for each type of toner and the results were found to be quite reproducible. 

Figure 3. Electron microscopic techniques used to study micromechanical processes in polymers (a) investigation of fracture surfaces by SEM (b) investigation by TEM of ultrathin sections prepared from deformed and selectively stained bulk material and (c) deformation of samples of different thicknesses (bulk, semithin, and ultrathin)9 using special tensile stages with SEM, HVEM, and TEM. The technique in (c) shows the possibility of conducting in situ deformation tests in the electron microscope.

Hashimoto H, Ishikawa H, Kusakabe M (1999) Preparation of whole mounts and thick sections for confocal microscopy. Methods Enzymol 307 84-107... 

The polyimlde molding compound prepared by any of the above techniques Is processed In a preheated mold at 550-600 F, followed by post cure to 625 F for 2-8 hours for development of maximum physical properties. Absence of volatile by-products permits facile production of sound moldings having thick sections, while the modest (1300) molecular weight of the prepolymer permits flow at reasonable temperatures. 

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