A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms. 2009

Irina Kolotuev, and Yannick Schwab, and Michel Labouesse
Department of Cell and Developmental Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire , 67400 Illkirch, France. ira.kolotueva@gmail.com

BACKGROUND CLEM (correlative live cell and electron microscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest). RESULTS We developed a CLEM technique that enables easy processing of small model animals and is adequate both for morphology and immunoelectron-microscopic specimen preparations. While this method has been initially developed for Caenorhabditis elegans samples, we found that it works equally well for Drosophila samples. It enables handling and observation of single animals of any complex genotype in real time, fixation by high-pressure freezing and flat embedding. Our major improvement has been the development of a precise mapping system that considerably simplifies and speeds up the retrospective location of the ROI within 1 mum distance. This method can be successfully used when correlative microscopy is required, as well as to facilitate the treatment of non-correlative TEM procedures. Our improvements open the possibility to treat statistically significant numbers of animals processed by electron microscopy and considerably simplifies electron-microscopic protocols, making them more accessible to a wider range of researchers. CONCLUSIONS We believe that this technique will contribute to correlative studies in multicellular models and will facilitate the time-demanding procedure of specimen preparation for any kind of TEM.

UI MeSH Term Description Entries
D008853 Microscopy The use of instrumentation and techniques for visualizing material and details that cannot be seen by the unaided eye. It is usually done by enlarging images, transmitted by light or electron beams, with optical or magnetic lenses that magnify the entire image field. With scanning microscopy, images are generated by collecting output from the specimen in a point-by-point fashion, on a magnified scale, as it is scanned by a narrow beam of light or electrons, a laser, a conductive probe, or a topographical probe. Compound Microscopy,Hand-Held Microscopy,Light Microscopy,Optical Microscopy,Simple Microscopy,Hand Held Microscopy,Microscopy, Compound,Microscopy, Hand-Held,Microscopy, Light,Microscopy, Optical,Microscopy, Simple
D008854 Microscopy, Electron Microscopy using an electron beam, instead of light, to visualize the sample, thereby allowing much greater magnification. The interactions of ELECTRONS with specimens are used to provide information about the fine structure of that specimen. In TRANSMISSION ELECTRON MICROSCOPY the reactions of the electrons that are transmitted through the specimen are imaged. In SCANNING ELECTRON MICROSCOPY an electron beam falls at a non-normal angle on the specimen and the image is derived from the reactions occurring above the plane of the specimen. Electron Microscopy
D003584 Cytological Techniques Methods used to study CELLS. Cytologic Technics,Cytological Technic,Cytological Technics,Cytological Technique,Technic, Cytological,Technics, Cytological,Technique, Cytological,Techniques, Cytological,Cytologic Technic,Technic, Cytologic,Technics, Cytologic
D004331 Drosophila melanogaster A species of fruit fly frequently used in genetics because of the large size of its chromosomes. D. melanogaster,Drosophila melanogasters,melanogaster, Drosophila
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D013997 Time Factors Elements of limited time intervals, contributing to particular results or situations. Time Series,Factor, Time,Time Factor
D016610 Tissue Embedding The technique of placing cells or tissue in a supporting medium so that thin sections can be cut using a microtome. The medium can be paraffin wax (PARAFFIN EMBEDDING) or plastics (PLASTIC EMBEDDING) such as epoxy resins. Embedding, Tissue
D016707 Tissue Fixation The technique of using FIXATIVES in the preparation of cytologic, histologic, or pathologic specimens for the purpose of maintaining the existing form and structure of all the constituent elements. Fixation, Tissue
D017173 Caenorhabditis elegans A species of nematode that is widely used in biological, biochemical, and genetic studies. Caenorhabditis elegan,elegan, Caenorhabditis

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