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Sci Rep. 2017 Oct 19;7(1):13583. doi: 10.1038/s41598-017-13348-6.

Microtome-integrated microscope system for high sensitivity tracking of in-resin fluorescence in blocks and ultrathin sections for correlative microscopy.

Lemercier N1,2,3,4, Middel V5, Hentsch D1,2,3,4, Taubert S1,2,3,4, Takamiya M5, Beil T5, Vonesch JL1,2,3,4, Baumbach T6, Schultz P7,8,9,10, Antony C1,2,3,4,5, Strähle U11.

Author information

1
Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1, rue Laurent Fries, 67404, Illkirch, France.
2
Centre National de la Recherche Scientifique, UMR7104, 1, rue Laurent Fries, 67404, Illkirch, France.
3
Institut National de la Santé et de la Recherche Médicale, U964, 1, rue Laurent Fries, 67404, Illkirch, France.
4
Université de Strasbourg, 1, rue Laurent Fries, 67404, Illkirch, France.
5
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany.
6
Laboratory for Applications of Synchrotron Radiation, Karlsruhe Institute of Technology, Kaiserstr. 12, 76131, Karlsruhe, Germany.
7
Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1, rue Laurent Fries, 67404, Illkirch, France. patrick.schultz@igbmc.fr.
8
Centre National de la Recherche Scientifique, UMR7104, 1, rue Laurent Fries, 67404, Illkirch, France. patrick.schultz@igbmc.fr.
9
Institut National de la Santé et de la Recherche Médicale, U964, 1, rue Laurent Fries, 67404, Illkirch, France. patrick.schultz@igbmc.fr.
10
Université de Strasbourg, 1, rue Laurent Fries, 67404, Illkirch, France. patrick.schultz@igbmc.fr.
11
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany. uwe.straehle@kit.edu.

Abstract

Many areas of biological research demand the combined use of different imaging modalities to cover a wide range of magnifications and measurements or to place fluorescent patterns into an ultrastructural context. A technically difficult problem is the efficient specimen transfer between different imaging modalities without losing the coordinates of the regions-of-interest (ROI). Here, we report a new and highly sensitive integrated system that combines a custom designed microscope with an ultramicrotome for in-resin-fluorescence detection in blocks, ribbons and sections on EM-grids. Although operating with long-distance lenses, this system achieves a very high light sensitivity. Our instrumental set-up and operating workflow are designed to investigate rare events in large tissue volumes. Applications range from studies of individual immune, stem and cancer cells to the investigation of non-uniform subcellular processes. As a use case, we present the ultrastructure of a single membrane repair patch on a muscle fiber in intact muscle in a whole animal context.

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