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. 2017 Oct 19;7:13583. doi: 10.1038/s41598-017-13348-6

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Microtome-Integrated Microscope (MIM). (a) The MIM is directly mounted on an UC7 Leica ultramicrotome and connected to the detector. The MIM and stereomicroscope are positioned at a right angle. The objective is facing the boat of the Jumbo knife to scan the ribbons for fluorescence detection. The MIM is composed of a high-sensitivity sCMOS Hamamatsu ORCA-Flash4.0 LT Camera (CA) installed on a customized optical tube tube (OT) equipped with a plan apochromatic lens and a dual-band fluorescence filter cube. The MIM can use various objective lenses (OBJ) as detailed in Table 1. (b) Model view of the MIM showing the structure of the optic tube with air objective and the connection to the fluorescence source on the side. (c) Optical scheme of the MIM showing the air objective (OBJ), the excitation (EX) and emission (EM) filters separated by the dichroic mirror (DI). Following the optical axis (OA), the light rays are focused on the sCMOS detector (CA) by a planapochromatic lens (L). The fluorescence source is reflected on the excitation filter (EX) by a mirror (M). (d) Side view of the MIM during sample processing, showing block-face imaging with the objective lens (OBJ) facing the Block (B) in the microtome sample holder. (e) Floating ribbon scanning with the objective lens above the knife boat (KB) during sectioning of the block (B) and with the diamond knife (DK). (f) “On-grid”- section imaging (G), also visible in inset with the same objective lens (OBJ).