reSolution_LNT_No1_en - Leica Microsystems

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BIOLOGY Perusing alternatives for automated staining of TEM thin sections Substitutes for Uranyl Acetate in TEM Thin Section Post-Staining Nicole Fellner1,3 , Marlene Brandstetter1,3 , Karin Trimmel1,2 , and Dr. Guenter P. Resch1,3 1IMP-IMBA-GMI Electron Microscopy Facility, Institute of Molecular Biotechnology, Vienna, Austria 2University of Applied Sciences, Wiener Neustadt, Austria 3Campus Science Support Facilities GmbH, Vienna, Austria Nicole Fellner (left), Marlene Brandstetter (in the middle of the front row), Günter Resch (in the middle of the back row), Harald Kotisch (right) 8 reSOLUTION Introduction Contrast in transmission electron microscopy (TEM) is mainly produced by electron scattering at the specimen: Structures that strongly scatter electrons are referred to as electron dense and appear as dark areas in the bright fi eld image, while structures which scatter fewer electrons appear bright (electron transparent) (Flegler et al., 1993). As electron scattering increases with atomic number, biological samples show hardly any inherent amplitude contrast in the TEM, as they are largely composed of light elements. To increase their contrast, electron dense stains can be added to the sample, the most commonly used heavy elements being: gold, platinum, tungsten, lead, and uranium. Biological specimens can be contrasted through various staining techniques: Particles like protein complexes or viruses can be embedded in heavy metal salts (negative staining), or the specimen can be covered with very thin electron-dense metal fi lms (replicas produced by shadowing). Cells and tissues can be infi ltrated with stain before embedding (Osmium tetroxide or uranyl acetate en bloc staining) or the ultra-thin sections are stained (Dykstra, 1992). The choice of reagents for the latter approach, called post-staining, is discussed in this article. The most frequently used method for post-staining is a twostep procedure of staining with uranyl acetate (UA), followed by lead citrate. Uranyl acetate is used as an aqueous or alcoholic solution with a pH for the saturated solution in the range of 3.5 to 4.0. The addition of alcohol, especially methanol, increases the solubility (Hayat, 2000). Uranyl acetate strongly stains proteins as well as nucleic acids and phospholipids. When applied after the uranyl acetate staining, lead citrate (prepared according to Reynolds, 1963) will increase this contrast (Dykstra, 1992). Staining can be performed either manually or automatically, both techniques have their advantages. For manual staining, a grid is fl oated, sectionside down, on a drop of a uranyl acetate solution for 10 minutes. After blotting off the stain, the grid is rinsed thoroughly with water to remove any residual unbound stain. This fi rst step is followed by 5 min. lead citrate staining, following the same procedure. The consumption of reagents is minimal, whereas the effort is relatively high. Alternatively, poststaining can be automated. This ensures increased reproducibility and time saving, though the amount of reagents used is higher. Using the automated contrasting device EM AC20 (Leica Microsystems, Vienna) allows for simultaneous staining of up to 20 grids per run with no effort and a guarantee for safety, both for the environment and the user. Although UA is an excellent and well characterized stain, replacements are sought for, for several reasons. When it needs to be handled as a powder, it is very toxic and carcinogenic if inhaled. Furthermore, also depleted uranyl acetate is considered a radioactive material, and hence subject to relevant regulations. Therefore, UA requires adequate storage and careful handling, which in turn increases cost for shipping and waste disposal. To minimize contact, the automated version with the AC20 is preferred by many users, in particular as readily prepared solutions are available, handling of solid UA can be avoided. Two reagents described in literature as replacements for UA caught our attention: oolong tea extract (OTE) and Platinum Blue. Only very little data is published about them and the methods are not well known in the EM community. Therefore, we have tested both with manual contrasting and for the fi rst time with the Leica EM AC20 instrument. To allow a direct comparison of results from the different post-staining techniques, the same sample was used for all tests: Liver tissue freshly dissected from mice was fi xed with 2.5% glutaraldehyde in 100 mmol/l Soerensen phosphate buffer and post-fi xed with 2.0% osmium tetroxide.
Pieces of tissue were dehydrated and embedded in Agar 100 epoxy resin and sectioned to a nominal thickness of 70 nm. Poststaining was performed as described below. Besides contrasting effi ciency and comparability of the results with UA, a number of other important parameters were assessed. Oolong Tea Extract OTE was described as post-stain to electron microscopy by Sato et al. (2003), and used in a small number of studies (Sato et al., 2008; Miller and Simakova, 2010). According to Rumpler et al. (2001), OTE is a type of half-fermented tea and produced as a foodstuff. Therefore it was assumed to be harmless for health and environment, even though the supplier failed to produce a material safety data sheet. It can be purchased from Ted Pella (http://www.tedpella. com), and is delivered as a powder. According to Sato et al. (2003), the polyphenolic components in OTE react with peptide bonds. A reaction with OTE and lead citrate in return, leads to an enhancement of the contrast. After tests with different OTE concentrations, 0.2% OTE dissolved in boiling ddH 2 O was used for further experiments, as already proposed by Sato et al. (2003). Compared to conventional staining with uranyl acetate, the manual application of OTE and the subsequent staining step with Reynold’s lead citrate was more time consuming (Table 1). Optimal results on the Leica EM AC20 were obtained also using an extended program at room temperature (Table 2). At a concentration of 0.2% OTE both manually as well as automatically stained samples show an increase in contrast even though it was clearly lower than with UA (Figs. 1 and 2). In our hands, contamination was observed more frequently than with UA, which mainly constituted a problem at lower magnifi cation. Despite extended washing steps, both with manual staining or the Leica EM AC20, this problem could not be eliminated. Platinum Blue Platinum Blue (Pt Blue) was used as an alternative to uranyl acetate in thin section post-staining by Inaga et al. (2007; 2009). This compound is a product of the reaction of cisdichlordiamineplatinum (II) with thymidine (Inaga et al., 2007). The reagent can be ordered directly from the Japanese producer Nisshin (http://nisshin-em.co.jp). Pt Blue is a hazardous material, which may cause eye irritation, cancer and effects on fertility as well as severe disorders of the bone marrow, kidneys and the nervous system (MSDS Nisshin). The commercially available 6% stock solution has been found to give good results at a dilution of 1:100 for the manual staining and 1:200 for the automated procedure. The incubation times for the manual staining method were the same as for UA staining (cf. Table 1). For staining using the Leica EM AC20, the step for Pt Blue were extended to 30 min, using the identical conditions as for UA. The grids were then rinsed with distilled water, stained with lead citrate, and washed as described in Table 2. Figures 1 and 2 illustrate that all organelles show good contrast and that the results are comparable in quality to pictures taken from sections stained with UA. Differences can be found in a more intensely stained mitochondrial matrix and a higher contrast of glycogen granules. The ribosomes appear weaker stained as compared to UA. Stain 1 Time Washing Stain 2 Time Washing UA 10 min 2 min ddH 2 O Lead citrate 5 min 2 min ddH 2 O OTE 25 min 6 min ddH 2 O Lead citrate 5 min 2 min ddH 2 O Pt Blue 10 min 2 min ddH 2 O Lead citrate 5 min 2 min ddH 2 O Table 1: Details of manual staining procedures Stain 1 Time Washing Stain 2 Time Washing BIOLOGY UA 30 min 2 min 20 sec ddH 2 O Lead citrate 7 min 2 min 20 sec ddH 2 O OTE 40 min 5 min ddH 2 O Lead citrate 7 min 2 min ddH 2 O Pt Blue 30 min 2 min 20 sec ddH 2 O Lead citrate 7 min 2 min 20 sec ddH 2 O Table 2: Automated staining procedures of the Leica EM AC20. The conditions for UA and lead citrate are suggested by Leica and can be found in the user's manual of the staining device. NANOTECHNOLOGY 9
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