A cryogenic, coincident fluorescence, electron and ion beam microscope

Boltje DB, Hoogenboom JP, Jakobi AJ, Jensen GJ, Jonker CTH, Kaag MJ, Koster AJ, Last MGF, de Agrela Pinto C, Plitzko JM, Raunser S, Tacke S, Wang Z, van der Wee EB, Wepf R, den Hoedt S. eLife (2022)


Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, a consortium with participation of the Raunser group presents in-situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: the team built a coincident 3-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing focused ion-beam scanning electron microscope. They show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope (TEM) tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

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