ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy

Abstract

In-resin fluorescence (IRF) protocols preserve fluorescent proteins in resin-embedded cells and tissues for correlative light and electron microscopy, aiding interpretation of macromolecular function within the complex cellular landscape. Dual-contrast IRF samples can be imaged in separate fluorescence and electron microscopes, or in dual-modality integrated microscopes for high resolution correlation of fluorophore to organelle. IRF samples also offer a unique opportunity to automate correlative imaging workflows. Here we present two new locator tools for finding and following fluorescent cells in IRF blocks, enabling future automation of correlative imaging. The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables ‘smart collection’ of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables ‘smart tracking’ of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes.

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

“…In recent years, huge progress has been made to tackle the challenge of correlating light and electron microscopy (CLEM) imaging 1 , 8 – 11 . However, these protocols are anything but routine yet 3 , 6 , 12 – 16 .…”

“…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. By having integrated this microscope directly onto the ultramicrotome, one can follow the ROI at each step from specimen localization in the block, block trimming, sectioning, tracking sections with fluorescence to documentation of landmarks and relative coordinates of the ROI.…”

Dedicated setup for the photoconversion of fluorescent dyes for functional electron microscopy

“…A number of new tools are being developed by commercial and academic research laboratories to simplify the CLEM workflow and to extend it to thick neuronal tissue. Some laboratories are developing new fluorescent proteins able to oxidise DAB in a light-independent way (Shu et al, 2011; Liss et al, 2015; de Beer et al, 2018), while others focus on new microscopes to improve the reliability of CLEM workflow (see Brama et al, 2016).…”

Serial block face scanning electron microscopy in cell biology: Applications and technology

“…This allows for the imaging of TEM sections via fluorescence microscopy; however this process is difficult to transfer to the block face EM techniques as the sections are not preserved during imaging and cannot be retrieved. The Collinson group has developed a small fluorescence microscope that clips to the knife of the 3view™and fits inside the microscope chamber in order that light microscope images can be collected alongside the EM data as the sections are cut (Brama et al, 2016). This mini-microscope is designed to be retrofitted to SBFSEMs and facilitates the collection of fluorescence microscopy images in between imaging/cutting cycles.…”