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.

Integrated light and electron microscopes (ILEMs) will enable a new generation of high-precision correlative imaging experiments. To fully exploit these systems, samples must contain dual-modality probes that highlight the position of macromolecules in the context of cell ultrastructure. We demonstrate that the fluorescent proteins (FPs) GFP (green), YFP (yellow) and mCherry can be used as dualmodality probes for ILEM when preserved using the inresin fluorescence (IRF) technique, which delivers stable active fluorophores in lightly stained, resin-embedded cells and tissues. However, we found that vacuum pressure in the ILEM affects the photophysics of FPs in IRF sections. Here, we show that reducing the vacuum pressure reduces fluorescence intensity of GFP and YFP, which is a consequence of water extraction from the sample and is reversible on recreation of partial pressure with water vapour (but not oxygen or nitrogen gas). We also find that, although fluorescence intensity is reduced at a partial pressure of 200 Pa (created using water vapour), the FP intensity is remarkably stable over time in vacuum and resistant to photobleaching during imaging. We are thus able to define imaging strategies for standard FPs acting as dual-modality probes in a single 'multi-colour' integrated microscope system.

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