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

Nicolas Lemercier,Didier Hentsch,Tanja Beil,Serge Taubert,Masanari Takamiya,Uwe Strähle,Claude Antony,Jean-Luc Vonesch,Tilo Baumbach,Patrick Schultz,Volker Middel

doi:10.1038/s41598-017-13348-6

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.

Highlights

A key challenge in biomedicine and biotechnology is to link the function of genes from their phenotypic effects in an organ and tissue context to the molecular events occurring at the subcellular level
A technically difficult and largely unsolved problem in correlating light and electron microscopy (CLEM) is the transfer of the same specimen between different imaging modalities without losing the ROI14,25,27
We report a new and highly sensitive microtome-integrated imaging system that combines a purpose-designed microscope mounted on a commercial ultramicrotome with a workflow (Direct-CLEM) optimized for efficient selection of plastic sections comprising the ROI

Summary

Introduction

A key challenge in biomedicine and biotechnology is to link the function of genes from their phenotypic effects in an organ and tissue context to the molecular events occurring at the subcellular level. A compact and highly sensitive epifluorescence microscope was developed and mounted onto an ultramicrotome to perform both fluorescence tracking and imaging on a single instrumental setup and at each step of the specimen preparation protocol While an another set-up was reported recently[11], our Microtome-Integrated-Microscope (MIM) is characterized by a remarkable sensitivity of in-resin detection of fluorescence by employing a new design of a lIght weight microscope optimized for photon collection. For the first time, we show here ultrastructural details of a macrophage interacting with a membrane repair patch These user cases demonstrate that the MIM is optimally suited to reliably detect single cellular and subcellular structures in large tissue volumes or in intact zebrafish embryos

Methods

Results

Conclusion