The NanoSIMS 50L instrument is a magnetic-sector, multi-collecting ion probe. The physical basis for ion micro-probe analysis is the ability to perform mass-spectrometry on secondary ions sputtered from a solid target by the continuous impact of a beam of charged particles. This primary beam sputters ionized atoms and small molecules (as well as many neutral particles) from the upper few nanometers of the sample surface. These secondary ions from the sample are transferred with high transmission to a high mass-resolution, multi-collection mass-spectrometer, where they are counted one-by-one in electron multiplier detectors, or as currents in Faraday cups.
The unique strength of the NanoSIMS ion microprobe is the ability to focus the primary beam (either Cs+ or O–) onto an extremely small spot on the sample surface, smaller than 100 nm in linear dimension. A controlled raster of this highly focused primary beam across the sample surface allows secondary ion images to be produced with a spatial resolution that can clearly resolve structures larger than a few hundred nanometers in linear dimension. For example, in recent studies we have been able to clearly resolve substructures of the cell nucleus, such as nucleolus and chromatin packages, along with individual clusters of glycogen in both liver and brain cells from mice (1, 2).
The conventional NanoSIMS instrument operates at room temperature and ultra-high vacuum (10-9-10-10 Torr). In order to preserve this vacuum, biological samples must be prepared to minimize volatilization or degassing. Classical sample preparation procedures developed for electron-beam imaging techniques (e.g. TEM and SEM) meet such constraints. However, these procedures involve steps such as fixation of the tissue with glutaraldehyde solutions, staining (e.g. with OsO4 or uranyl-acetate), dehydration in ethanol series, and finally embedding into epoxy resin. These procedures effectively remove soluble compounds originally present in the tissue. Left behind in the sample are macromolecular structures, such as proteins, lipids, RNA, and DNA. These macromolecular structures can, on the other hand, be isotopically imaged in great detail with a conventional NanoSIMS instrument at a spatial resolution down to 100-50 nm. This has already created vigorous research programs and important biological insights have been gained across an impressive range of organisms; reviewed in ref. (3).
However, a multitude of fundamental biological processes involve the action of soluble compounds (ions, metabolites, drugs, etc.) that cannot be imaged with the conventional NanoSIMS instrument because they are lost or significantly displaced during classical sample preparation. The only certain way to preserve and observe soluble molecular compounds and ions unperturbed in situ in a biological tissue is to create and maintain highly controlled cryo-conditions throughout the chain of preparative and observational procedures. Our method is based on state-of-the-art cryo-methods for sample preparation (starting with high-pressure freezing, followed by cryo-planing of the tissue in a cryo-ultramicrotome) and subsequent ultra-structural (i.e. sub-cellular) observations with cryo-scanning electron microscopy (4). What is missing from the currently existing observational chain is an instrument that can isotopically image cryo-prepared samples with ultra-high spatial resolution, permitting precise correlation with the structural information provided by electron microscopy. Our vision has been to develop a CryoNanoSIMS to achieve this goal. From an instrument development point of view, we have now succeeded in this and we will present our preliminary data.
1 Takado, Y. et al (2014). Nanomed Nanotech Biol Med, doi: 10.1016/j.nano.2014.09.007.
2 Takado, Y. et al (2015). J Chem Neuroanat 69, 7-15, doi:10.1016/j.jchemneu.2015.09.003.
3 Hoppe, P. et al (2013). Geostand Geoanal Res 37, 111-154, doi:10.1111/j.1751-908X.2013.00239.x.
4 Walther, P. & Müller, M. (1999). J Microsc 196, 279-287, doi: 10.1046/j.1365-2818.1999.00595.x.
To cite this abstract:Louise Helene Søgaard Jensen, Tian Cheng, Florent Olivier Vivien Plane, Stéphane Escrig, Arnaud Comment, Ben van den Brandt, Bruno Martin Humbel, Anders Meibom; En route to ion microprobe analysis of soluble compounds at the single cell level: The CryoNanoSIMS. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/en-route-to-ion-microprobe-analysis-of-soluble-compounds-at-the-single-cell-level-the-cryonanosims/. Accessed: September 18, 2020
EMC Abstracts - https://emc-proceedings.com/abstract/en-route-to-ion-microprobe-analysis-of-soluble-compounds-at-the-single-cell-level-the-cryonanosims/