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cryo electron microscopy, cryo-electron tomography • 3 min reading time

Cryo R&D story, episode 5: challenges in combining light microscopy inside a FIB/SEM chamber

The fifth episode of the Delmic Cryo R&D story is out now! Read the blog story of our Experimental Physicist Marre Niessen to see how the development team designed the optical module of our ENZEL system.

I am Marre Niessen and am working at Delmic as an Experimental Physicist. Within our cryo department, I mainly work on the ENZEL system. I test new designs made by our mechanical team to validate new functionalities and indicate improvements.

In the ENZEL system, light and electron microscopy are combined to generate lamella for cryo-ET in a targeted manner. Light microscopy is widely used in biological research to study specific proteins, their relationships, and their functionality. Proteins of interest are labelled with fluorescence for visualization. Multiple fluorescent labels can be combined to localise regions of interest to study the interaction of proteins in the cell. In addition, biological samples are studied with cryo-electron microscopy revealing structural information. 
As Daan explained in episode 2 of this Delmic Cryo R&D story series, ENZEL adds light microscopy capabilities to a dual-beam FIB/SEM system to simplify sample processing for cryo-ET. The light microscope guides the milling of biological samples by visualizing the region of interest. In this post, I will highlight the design of the optical module, which contains the light microscope of ENZEL. 

In the FIB-SEM, the sample is located at the intersection of the SEM and FIB focal points. The optical system of ENZEL has to coincide with that intersection. We have tackled three design challenges to integrate the different microscopes for targeted milling: limited space for an optical microscope, adaptable position of the three-beam alignment and 3D-localisation of the region of interest. 

First, the optical path should be inside the vacuum chamber, which Delmic already achieved in the SECOM system. For ENZEL, the challenge lay in the space requirement of a light microscope which is limited by the SEM chamber size. Our innovative solution was found in a drawer-like system. The optical elements are all tightly packed in a small cassette. This cassette can slide into the chamber where a window enables a free optical path to the sample. Resulting in a full light microscope integrated into the vacuum chamber.  

Second, the position of the objective lens should be adaptable to multiple FIB/SEM systems, where the intersections of SEM and FIB have different positions. The adaptable position is also used for the alignment of the three beams. Our solution is to have both the sample, as well as the objective lens, on a movable stage. The two stages can align the sample and the objective lens with the intersection of the FIB/SEM, which is critical for targeted milling. 

Finally, we tackled the challenge of the 3D localization of the target protein. 3D-localization enables the inclusion of the target in the prepared lamella. As the FIB mills at a tilted angle, the localization of the target in the z-axes has to be accurate enough to include the ROI in the 150 nm - 250 nm thick lamella. The addition of the 3D localization will make the life of our customers easier, as there is a higher chance that the lamella made will contain the protein of interest.  

Our solutions for the optical module, together with the innovations discussed before by Daan, Keith and Caspar make ENZEL a desirable solution for FIB/SEM users with cryo-ET ambition. The addition of a micro cooler, a transfer system and a tilted objective with corresponding light microscopy capabilities enable targeted milling of lamellae in cryogenic samples. We hope that these R&D stories provide insight into our development process and we are looking forward to seeing our work put into action by many future customers.
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Marre Niessen