Understanding the biological role of molecules requires to know their precise localization and structural environment. Thanks to fluorescence microscopy and biotechnologies, the localization of proteins of interest has become relatively easy. But in more and more cases, this resolution is not enough and the lack of data on the molecule/cell environment does not allow the characterization of their biological function. Transmission Electron Microscopy (TEM) and immuno-gold labelling can be used to characterize at the same time the cellular compartments associated with the protein of interest and the ultrastructure of the cellular environment at a high resolution. However, the antigenicity preservation as well as the antibody production are sometimes difficult. Moreover, TEM observations only give access to a static “snapshot” of a fixed, dead sample. For several years, many attempts have been made to correlate Fluorescent and Electron Microscopy in order to combine the advantages of both microscopy techniques on a unique sample. In this way, we develop on the BIC new protocols of sample preparation for animal and vegetal sample (culture cells and tissues), allowing to maintain simultaneously the fluorescence and the overall ultrastructure (Figure 1 and 2), using Quick Freeze Substitution (McDonald KL and Webb RI, J. Microsc, 2011). This technique will allow the correlation of both Light and Electron Microscopy data on the same section (CLEM: Correlative Light Electron Microscopy), works with classical fluorescent tags and improve the antigenicity for gold immunolabelling.

Acknowledgement: We acknowledge France BioImaging, IDEX Bordeaux and the CNRS for the support.

Plant lines and reconstructed tissue are respectively friendly given by Claire Bréhélin (UMR5200, Laboratoire de Biogénèse membranaire, Bordeaux) and, Murielle Rémy and Olivia Kérourédan (INSERM U1026 BioTis, Université de Bordeaux)

Figures:

Fig 1 : In resin fluorescence of tissu reconstruction containing epithelial cells tagged with tomato fluorophore. Observation on ultrathin section, in DIC (A) and in TRITC (B) channel with a wildfield microscope. The ROI (arrow)(C) was then observed under TEM on the same section (x20000).

Fig 2: In resin fluorescence of root with a green fluorescent tag. Live root expressing a fluorescent tag observed by fluorescence microscopy (A). Fluorescent Tag (green) and nucleus staining (blue) observed on ultrathin section (B). Mapping of green fluorescent spots based on nucleus pattern in the same section by TEM (C).

« Back to The 16th European Microscopy Congress 2016

EMC Abstracts - https://emc-proceedings.com/abstract/correlative-microscopy-on-animal-and-vegetal-samples-with-in-resin-fluorescence-approach/