Comparison of different preparation methods of biological samples

Comparison of different preparation methods of biological samples

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Journal of Microscopy, Vol. 233, Pt 2 2009, pp. 309–319 Received 8 November 2007; accepted 5 October 2008 Comparison of different preparation methods of biological samples for FIB milling and SEM investigation V. LE ˇ SER ,D .DROBNE , ˇ Z. PIPAN ,M .MILANI & F. TATTI Department of Biology, University of Ljubljana, Veˇcna pot 111, SI-1000 Ljubljana, Slovenia Materials Science Department and Laboratory FIB/SEM “Bombay “, University of Milano-Bicocca, Via Cozzi 53, I-20125 Milano, Italy FEI Italia, Viale Bianca Maria 21, I-20122 Milano, Italy Key words. Chemical fixatives, conductive staining, Crustaceans, digestive gland epithelium, Porcellio scaber , terrestrial isopod. Summary When a new approach in microscopy is introduced, broad interest is attracted only when the sample preparation procedure is elaborated and the results compared with the outcome of the existing methods. In the work presented here we tested different preparation procedures for focused ion beam (FIB) milling and scanning electron microscopy (SEM) of biological samples. The digestive gland epithelium of a terrestrial crustacean was prepared in a parallel for FIB/SEM andtransmissionelectronmicroscope(TEM).Allsampleswere aldehyde-fixed but followed by different further preparation steps. The results demonstrate that the FIB/SEM samples prepared for conventional scanning electron microscopy (dried) is suited for characterization of those intracellular morphological features, which have membranous/lamellar appearance and structures with composition of different density as the rest of the cell. The FIB/SEM of dried samples did not allow unambiguous recognition of cellular organelles. However, cellular organelles can be recognized by FIB/SEM when samples are embedded in plastic as for TEM and imaged by backscattered electrons. The best results in terms of topographical contrast on FIB milled dried samples were obtained when samples were aldehyde- fixed and conductively stained with the OTOTO method (osmium tetroxide/thiocarbohydrazide/osmium tetroxide/ thiocarbohydrazide/osmium tetroxide). In the work presented here we provide evidence that FIB/SEM enables both, detailed recognition of cell ultrastructure, when samples are plastic embedded as for TEM or investigation of sample surface morphology and subcellular composition, when samples are dried as for conventional SEM. Correspondence to: Damjana Drobne. Tel: + 386 1 423 33 88; fax: + 38612573 390; e-mail: [email protected] Introduction First evidence of a successful application of the focused ion beam (FIB) / scanning electron microscope (SEM) for biologicalsampleswaspublishedin1993(Young et al. ,1993). Within a decade, a bunch of papers appeared reporting the applicability of FIB/SEM in exposing and investigating the subcellular structures of a variety of biological samples (see Milani et al. , 2006; Hayles et al., 2007). Despite the potential advantages of FIB/SEM, it is still not accepted as a method of choice in biological structure research at submicrometre scale. A great deal of this restraint against the application
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This note was uploaded on 05/28/2010 for the course BIOLOGY 03234 taught by Professor Sochacka during the Spring '10 term at Ghent University.

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Comparison of different preparation methods of biological samples

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