New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography

Abstract

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens.

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

“…Like immuno-EM, room-temperature CLEM also requires chemically fixed and dehydrated cells, which can distort or obscure important structural features (Lucic et al, 2013, Afzelius and Maunsbach, 2004). Nevertheless, room-temperature CLEM has been instrumental in the visualization of numerous bacterial and mammalian cellular events that would otherwise have been challenging or impossible to capture prior to the advent of this method (Grabenbauer et al, 2005, Muller-Reichert et al, 2007, Bertipaglia et al, 2016, Darcy et al, 2006, Schorb et al, 2016, Avinoam et al, 2015, Kukulski et al, 2011, Kukulski et al, 2012, Redemann and Muller-Reichert, 2013, Kapoor et al, 2006). …”

“…To visualize fluorescence inside frozen-hydrated cells, cryogenic LM (cryo-LM) stages are used (Schlimpert et al, 2012, Bertipaglia et al, 2016, Schorb and Briggs, 2014, Briegel et al, 2010, Schellenberger et al, 2014, Schorb et al, 2016). Unfortunately, because the sample has to be kept frozen, long-working-distance air-objective lenses with low numerical apertures are used instead of oil-immersion lenses.…”

“…Cryo-CLEM experiments on mammalian cells have been previously performed using organic fluorescent dyes or proteins tagged with fluorophores (Kaufmann et al, 2014, Liu et al, 2015, Schorb and Briggs, 2014, Schorb et al, 2016, Bykov et al, 2016, Schellenberger et al, 2014, Jun et al, 2011). Though organic dyes are very bright, it can be challenging to use these compounds to efficiently label specific proteins inside cells.…”

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

“…Alternatively, an integrated light and electron microscope can be used, which combines both cryo-fLM and cryo-EM imaging sources in the column of the TEM 45 . The Leica cryo-CLEM system 46 has the advantage of using a unique ceramic-tipped, short-working-distance objective with a resolution limit of ~200 nm. Our decision to use the Leica cryo-CLEM system was also driven by the seamless nature of the hardware and software, which facilitates the direct registration of coordinates between the two imaging platforms and eliminates time-consuming generation of LM–TEM overlay images before collecting high- resolution cryo-ET data.…”

Correlated Cryo Super‐Resolution Light and Cryo‐Electron Microscopy on Mammalian Cells Expressing the Fluorescent Protein rsEGFP2

“…Biological samples are typically prepared for CLEM using chemical fixatives such as paraformaldehyde, which are known to introduce artefacts . In contrast, nonchemical vitrification, i.e., cryo‐fixation, enables investigation of biological samples in an unperturbed, near‐native state, and allows cryoCLEM to be utilized . Generally, (cryo)FM is used to locate regions of interest, such as the location of labeled biomolecules, exploiting the large field of view provided by (cryo)FM.…”