We present a novel optical superresolution (SR) technique using integrated correlative light and electron microscopy. Recent advances in SR techniques has revolutionized the field of optical microscopy by achieving image resolutions well below the diffraction limit, the fundamental resolution limit of traditional optical microscopy. Current SR methods involve stochastic techniques, beam-shaping in combination with confocal scanning, external control over excited state relaxation pathways, and/or structured illumination . Correlation of SR data with ultrastructural images obtained with electron microscopy (EM) has been demonstrated , but requirements for SR microscopy are often in conflict with those for EM. Moreover, the optical localization accuracy in the correlation image may be severely compromised compared to the SR resolution by the additional error introduced in aligning the separate SR and EM images. Here, we demonstrate a novel approach for correlative SR-EM using a focused electron beam to locally modify the fluorescence signal of fluorophores, and detecting the change in fluorescence intensity with a wide-field epi-fluorescence microscope.
We use an integrated light-electron microscope  that is used for correlative light and electron microscopy (CLEM) . The integrated light microscope allows us to record the fluorescence signal while scanning the electron beam through the optical field of view. By correlating changes in the fluorescence decay with the instantaneous electron beam position and the other EM signals, we obtain a SR fluorescence image (Fig.1). This SR fluorescence image is in perfect registry with the simultaneously acquired EM image.
In first experiments on rat pancreas tissue, immuno-labelled for insulin and guanine quadruplexes using different Alexa Fluor dyes, we have achieved a lateral resolution below 100nm (Fig. 2). We will discuss further implementation of our technique towards higher resolution, paving the way towards precise localization, within the EM ultrastructure, of bio-molecules labelled with standard fluorescent dyes.
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To cite this abstract:Lennard M. Voortman , Aditi Srinivasa Raja, Aaro Väkeväinen, Pascal de Boer, Ben N.G. Giepmans, Pieter Kruit, Jacob P. Hoogenboom; Electron-beam induced fluorescence superresolution with 100nm resolution in CLEM on labelled tissue sections. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/electron-beam-induced-fluorescence-superresolution-with-100nm-resolution-in-clem-on-labelled-tissue-sections/. Accessed: January 18, 2020
EMC Abstracts - https://emc-proceedings.com/abstract/electron-beam-induced-fluorescence-superresolution-with-100nm-resolution-in-clem-on-labelled-tissue-sections/