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.
Highlights
Correlative light and electron microscopy (CLEM) workflows are widely used in biomedical research (Muller-Reichert & Verkade, 2012; Muller-Reichert & Verkade, 2014)
Our previous work demonstrated that imaging In-resin fluorescence (IRF) sections with an integrated light and scanning electron microscope (ILSEM) results in high precision correlation of fluorescent protein (FP) signal to cell organelles (Peddie et al, 2014a)
Design requirements for the ultraLM The ultraLM design took into account the following design principles: the microscope must be positioned in front of the resin for imaging; at a safe distance above the diamond knife to avoid damage; without obscuring the operator view of the sections in the boat; whilst allowing access to the boat for section retrieval; with a resolution of at least 1 μm for subcellular feature recognition, and a field of view of at least 500 × 500 μm, to view most or all of the resin blockface; without adding vibration into the system
Summary
Correlative light and electron microscopy (CLEM) workflows are widely used in biomedical research (Muller-Reichert & Verkade, 2012; Muller-Reichert & Verkade, 2014). Recent CLEM workflows preserve the fluorophores during resin-embedding, enabling post-embedding FM and EM on the same sample (Bell et al, 2013; Johnson et al, 2015; Kukulski et al, 2011; Nixon et al, 2009; Peddie et al, 2014a; Watanabe et al, 2011) These in-resin fluorescence (IRF) blocks can be cut using an ultramicrotome, to produce ultrathin IRF sections of 50–200 nm, which can be imaged sequentially with stand-alone fluorescence and electron microscopes (Figure 1B), or in situ in an integrated light and electron microscope (Figure 1C) (Peddie et al, 2014b). Our previous work demonstrated that imaging IRF sections with an integrated light and scanning electron microscope (ILSEM) results in high precision correlation of fluorescent protein (FP) signal to cell organelles (Peddie et al, 2014a)
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