Towards multimodal in situ cryo-FIB-SIMS imaging in biological systems

Abstract text (incl. figure legends and references)

Introduction: One of the struggles of correlative multimodal imaging in biological systems is the necessity for utilizing several analytical instruments to permit high-resolution and high-sensitivity morphological and chemical analyses. This induces time-consuming sample transfers and subsequent data processing and correlation. For cryo-investigations, not only is there a limited availability of cryo-compatible instruments, but there is the additional concern of contaminating or even damaging the frozen-hydrated sample during sample-transfer due to ambient temperature and humidity.

Our group specializes in incorporating several complimentary techniques into one individual instrument to streamline such correlative analytical workflows, all the while focusing on the vital key characteristics, i.e. high spatial resolution, chemical sensitivity, dynamic range (for the detection and mapping of atoms and small clusters with concentrations varying over several orders of magnitude) and isotopic selectivity.

Objectives: We present a novel instrument devised for multimodal in situ imaging and chemical analysis of biological samples in a close to native cryogenic state.

Materials & Methods: Previously, we described the in-house developed npSCOPE instrument [1], based on a Gas Field Ion Source using finely focused Helium and Neon ion beams and capable of performing: a) Secondary Electron (SE) imaging (providing morphological and topographical information; lateral resolution: < 1 nm); b) Scanning Transmission Ion Microscopy (STIM, providing bright and dark field imaging; lateral resolution: < 6 nm); and c) Secondary Ion Mass Spectrometry (SIMS, employing a compact high-performance double-focussing magnetic sector mass spectrometer equipped with a continuous focal plane detector, allowing a full mass spectrum to be recorded for each single pixel; lateral resolution: < 15 nm).

Here, we describe new developments that allow the investigation of frozen-hydrated biological samples at temperatures < -140° C close to their native state on the npSCOPE under high vacuum conditions. To achieve this, we equipped the instrument with a custom-built cryogenic sample stage, and we developed a customized cryo-sample transfer system structured around a prototype cryogenic-glovebox, ensuring optimal environmental conditions during sample handling.

Results: We present proof-of-principle results comparing data obtained on various chemically fixed room temperature (RT)-compatible samples which were investigated both at RT and at < -140° C that show that all three imaging modes function artefact-free at cryogenic temperatures, as well as early results obtained from frozen-hydrated samples.

Conclusion: The here presented cryo-workflow on the npSCOPE will allow the chemical analysis of biological samples from various fields of research under close to native conditions.

Funding: This project has received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 720964 and by the Luxembourg National Research Fund via the project INTER/DFG/19/13992454.

References:

[1] O. De Castro et al., "npSCOPE: A New Multimodal Instrument for In Situ Correlative Analysis of Nanoparticles," Anal. Chem., vol. 93, no. 43, pp. 14417–14424, Nov. 2021, doi: 10.1021/acs.analchem.1c02337.