Integrated Cryo-Correlative Microscopy


Discussion We have shown how an integrated FLM can be used to


guide on-the-grid lamella milling. Since METEOR is a flexible system, we expect that it can be applied to a wide range of other workflows and applications. Automated lamella milling. In recent years, several have


methods been developed to automate lamella


milling [22–24]. Tese methods significantly decrease the user interaction time and increase the throughput of the microscope. It would be advantageous if in this process the ROIs could be selected automatically based on FLM images. Tis would allow automatic creation of lamellae that include rare events that can only be captured using FLM. An integrated FLM provides this opportunity since the images are captured using the same sample stage. Moreover, it provides the opportunity to include an automatic check aſter milling to confirm the ROI is present in the lamella and it is worthwhile to image in the cryo-TEM. High-pressure frozen samples. We have shown how an


integrated cryo-CLEM system can streamline the on-the-grid lamella workflow. Tis workflow is limited to small cells, while studying the biomolecular interaction in tissue and multi- cellular organisms can give even more physiologically relevant information. To process these thick samples, additional steps are required such as liſting out a chunk of material [25–27]. Tese workflows are more error prone, and finding the ROI is even harder [28]. Terefore, the application of an integrated FLM to streamline this process will be very beneficial. Super resolution. Currently METEOR provides FLM


imaging at diffraction-limited resolutions using a LED light source and widefield illumination. Breaking the diffraction limit barrier can improve the lamella targeting accuracy, as well as bridge the resolution gaps between the FLM and TEM data. Several cryo-super-resolution FLM methods have already been described in the literature and demonstrated various resolution-enhancing factors [29–31]. Te next challenge will be to develop an integrated cryo-super-resolution system.


Conclusion We have shown how an integrated FLM system such as


METEOR can be used effectively to study biological processes with correlative FIB-milling and cryo-ET. We have demon- strated that METEOR can be used to accurately target ROIs for lamella milling. Moreover, it is easy to reinspect the sample with FLM aſter the milling process, confirming the ROI is present in the lamella and thereby improving the sample yield. We also showed that imaging a thin lamella with an integrated FLM does not cause devitrification or damage to the structure of interest.


Acknowledgements Te yeast strain was provided by Florian Wilfling (Max


Planck Institute for Biophysics, Frankfurt, Germany). Anna Bieber was supported by a Boehringer Ingelheim Fonds PhD fellowship. Te METEOR development work is supported by the European SME2 grant number 879673 – Cryo-SECOM Workflow.


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