Use of cryo-negative staining in tomographic reconstruction of biological objects: application to T4 bacteriophage |
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Authors: | Messaoudi Cedric Boudier Thomas Lechaire Jean-Pierre Rigaud Jean-Louis Delacroix Hervé Gaill Françoise Marco Sergio |
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Institution: | Institut Curie section recherche, UMR-CNRS 168 et LRC-CEA 34V. 11, rue Pierre et Marie Curie, 75231 Paris 05, France. |
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Abstract: | Recent advances in electron microscopy and image analysis techniques have resulted in the development of tomography, which makes possible the study of structures neither accessible to X-ray crystallography nor nuclear magnetic resonance. However, the use of tomography to study biological structures, ranging from 100 to 500 nm, requires developments in sample preparation and image analysis. Indeed, cryo-electron tomography present two major drawbacks: the low contrast of recorded images and the sample radiation damage. In the present work we have tested, on T4 bacteriophage samples, the use of a new preparation technique, cryo-negative staining, which reduces the radiation damage while preserving a high signal-to-noise ratio. Our results demonstrate that the combination of cryo-negative staining in tomography with standard cryo-microscopy and single particle analysis results in a methodological approach that could be useful in the study of biological structures ranging in the T4 bacteriophage size. |
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Keywords: | Electron tomography Cryo-microscopy Cryo-negative staining 3D reconstruction T4 bacteriophage |
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