cryo electron microscopy, cryo-electron tomography • 3

METEOR: an integrated top down cryo-CLEM imaging system

METEOR (fluorescent light microscope) image of a 1 μm thick lamella of yeast overexpressing eGFP-Ede1 with the SEM image overlaid. Images courtesy of A. Bieber, C. Capitanio and O. Schioetz (MPI Biochemistry)

In our previous blogposts we discussed different applications of cryogenic electron tomography (cryo-ET) and how an integrated fluorescent light microscope (FLM) could benefit this research. In this blogpost we will dive deeper into the Delmic’s new integrated FLM system METEOR and explain how it can help simplify your research. 

Highly optimisable system

METEOR is an FLM that is integrated in a (cryo) focused ion beam (FIB)/ scanning electron microscope (SEM) chamber. METEOR is positioned on existing ports of the FIB/SEM chamber ensuring easy installation without permanent changes to your FIB/SEM system. Due to the modular design METEOR can be optimized for your research. The emission filters are customisable to best match the emission peaks of your fluorescence labels in cryogenic conditions. Moreover, the objective, camera and excitation filters can be adjusted according to your wishes.

Open source and user friendly software

METEOR is accompanied by the powerful open-source acquisition software ODEMIS, which has features such as acquisition of z-stacks, multi-channel imaging and large area mapping. ODEMIS controls the stage of the FIB/SEM through an Xt adapter for seamless switching from FIB milling or SEM imaging to FLM imaging. For advanced users, a scripting interface in Python gives you full control of the hardware and the imaging algorithms.

Simplify your workflow 

Having an FLM inside the FIB/SEM will greatly reduce the amount of handling steps required, resulting in less contamination, less damage and a more efficient cryo-ET workflow. Additionally, switching between FLM and SEM imaging without transferring the sample allows direct correlation of the SEM and FLM images. This significantly speeds up identification of the region of interest (ROI) and ensures the milling process is performed at the correct site and does not endanger the structure of interest. Furthermore, it is easy to inspect the sample with FLM during the milling process, thereby improving the quality of the samples that ultimately end up in the cryo-TEM. 

Widely applicable system

METEOR is a flexible system that can be applied to a wide range of research areas and workflows. METEOR can be integrated in the standard on-grid-milling cryo-ET workflow and enhance research areas such as virology, membrane trafficking and neurobiology. 

In addition, METEOR could be integrated in the newly developed automated milling software applications. The ROIs could be selected based on the FLM image taken by METEOR. After the different rounds of milling the stage automatically moves to the METEOR imaging position and where FLM images are taken. Lamellae where the ROI is already milled away can be discarded to optimize valuable cryo-TEM time. 

Furthermore, METEOR could also simplify the cryo-FIB lift-out workflow, a very promising technique that allows the study of in situ structures in multi cellular organisms or tissues. The success rate is now smaller than 20%. The integration of METEOR with an FLM will reduce the number of transfer steps and thus the surface contamination which will lead to a higher yield. Moreover, having an FLM inside the FIB/SEM chamber allows FLM imaging of the extracted volume to guide the final FIB milling step. 

Image above: METEOR (fluorescent light microscope) image of a 1 μm thick lamella of yeast overexpressing eGFP-Ede1 with the SEM image overlaid. Images courtesy of A. Bieber, C. Capitanio and O. Schioetz (MPI Biochemistry)

As described, METEOR is a powerful system that is easily adaptable to your workflow and application area. Do you want to learn more about METEOR and its features? Sign up for the online launch demonstration on the 28th or 29th of October. 

This work is supported by the Eurostars grant № E13008 - CETFlow
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Marit Smeets