Confocal Laser Scanning Microscopy and Two Photon Excitation Microscopy as Tools to Study Testate Amoebae Z. Burdíková * ** , M. Čapek * **** , P. Ostašov * , E.A.D. Mitchell ***** , J. Machač *** , L. Kubínová * * Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, Prague 4 - Krč, 14220 Czech Republic ** Institute of Geology and Palaeontology, Faculty of Science, Charles university, Albertov 6, Prague, 128 43 Czech Republic *** Institute of Botany, Academy of Sciences of the Czech Republic, v.v.i., Lesní 322, Průhonice, 252 43 Czech Republic **** Faculty of Biomedical Engineering, Czech Technical University in Prague, nám. Sítná 3105, Kladno 2, 272 01 Czech Republic ***** WSL Swiss Federal Research Institute, Wetlands Research Group, Lausanne, Switzerland Testate amoebae (TA) (Protozoa: Rhizopoda) are a group of unicellular animals (20-400 µm) protected by a test. The test encloses the cell plasma and has usually a single aperture for the pseudopodia. A proteinaceous organic matrix is the basic test component. There are four main test types: proteinaceous (species with a flexible or rigid test), calcareous, siliceous (species which secrete their own regular siliceous test plates, so-called idiosomes) and agglutinated (species which include extraneous mineral particles in their test structure, so-called xenosomes). TA are commonly used as model organisms in population ecology, ecotoxicology and paleoecology, for their cosmopolitan dispersion and species-specific ecological preferences together with low ecological valence to changes of environmental surroundings [1]. TA play a very important role in most aquatic and terrestrial ecosystems and are, therefore, an essential subject of many scientific studies, such as paleoecological, environmental, etc. [2]. However, only restricted number of articles was published about their morphology, biometry and ecological preferences [3]. TA classification is based on the detailed study of structure of the test. The cytoplasm, the types of pseudopodia and nuclei for the majority of these species have not been yet observed and their visualisation will help with systematic identification. In many of TA-based ecological studies it is important to know precise estimates of test volumes. The volume of biomass inside the tests can then be calculated with the help of appropriate conversion factor. The volume of biomass is usually evaluated using simple measurements and using geometrical shapes of TA tests. This approach may not be accurate. The most common technique used for studying TA is conventional wide-field optical microscopy, more specifically bright-field and phase contrast microscopy. However, data produced by this technique have low image contrast. Generally, wide-field methods are not powerful enough to distinguish important features of the test or the inner structures of the cell. More convenient and widely used tool is electron microscopy: scanning electron microscopy (SEM) and environmental SEM (ESEM). By using these techniques it is possible to examine the surface features of TA with famously high resolution, reasonably good depth of field and to perform quantitative elemental analyses of shell structures. However, SEM and ESEM cannot be used to examine living TA or internal structures of the amoeba. Moreover, for species determination using 1142 doi:10.1017/S1431927610060897 Microsc. Microanal. 16 (Suppl 2), 2010 © Microscopy Society of America 2010 https://doi.org/10.1017/S1431927610060897 Downloaded from https://www.cambridge.org/core. IP address: 3.236.55.199, on 11 Jun 2020 at 03:13:20, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms.