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PROTOCOL

Preparative Techniques for Transmission Electron Microscopy and Confocal laser Scanning Microscopy of lichens ASUNCI6N DE LOS

Rios

and CARMEN ASCASO

Introduction Transmission electron microscopy

Ultrastructural study of lichen symbionts can provide valuable information about symbiotic performance complementary to that obtained using other techniques (Lallemant et al. 1986). Transmission Electron Microscopy (TEM) was first used to describe the cellular organelles of both symbionts (Jacobs and Ahmadjian 1969; Boissiere 1972; Galun et al. 1970, 1974; Peveling 1973, 1974, 1976; Ascaso and Galvan 1975, 1976). Later, different aspects oflichen symbiosis were studied, for example cellular membranes and cell wall with the freeze-etching electron microscopy technique (Ellis and Brown 1972; Peveling and Robenek 1980; Ascaso et al. 1985; Honegger 1986a; Rapsch et al. 1986). TEM has contributed to the understanding of different types of mycobiont-photobiont relationships in lichens, e.g. by observing the physical contacts between symbionts (for reviews see Honegger 1984, 1985, 1986b). The study of storage bodies in both symbionts provides indirect information on biotrophic relationships (Valladares and Ascaso 1994). Some authors have described the variability of lichen ultrastructure in relation to season or environment (Holopainen 1982; Scott and Larson 1986; Fiechter and Honegger 1988; Balaguer et al. 1999). In some investigations, TEM techniques have revealed structural changes due to different experimental conditions, ranging from desiccation to environmental pollution (Eversman and Sigal 1984, 1987; Ascaso et al. 1986, 1988; Brown et al. 1987, 1988; Balaguer et al. 1996, 1997; Tarhanen et al. 1997). Asunci6n de los Rios, Centro de Ciencias Medioambientales (CSIC), Serrano 115 dpdo, Madrid, 28006, Spain (phone + fax 34-915640800; e-mail [email protected]) Carmen Ascaso, Centro de Ciencias Medioambientales (CSIC), Serrano 115 dpdo, Madrid, 28006, Spain

[S!;l

I. C. Kranner et al. (eds .), Protocols in Lichenology © Springer-Verlag Berlin Heidelberg 2002

88

ASUNCION DE LOS

Rios

and CARMEN ASCASO

As poikilohydric organisms, lichens have no means of controlling their water relations, and they can spend long periods in a desiccated state. Therefore it is important not only to know the ultrastructure of hydrated thalli but also the ultrastructure of desiccated thalli. However, conventional techniques for TEM involve immediate rehydration of the thalli, which implies that it is impossible to study all of the morphological and ultrastructural details of lichens in the desiccated state (De los Rios et al. 1999). Cryotechniques have been used to overcome this problem (Honegger and Peter 1994; Honegger et al. 1996) but their application in the preparation of lichen thalli for TEM presents many methodological problems. Cryotechniques rely on low temperatures to ftx and stabilize specimen ultrastructure, which should then reflect the living state accurately. Optimising TEM cryotechniques in lichens is an important