link to SIMS on first page, cameca

An updated list of NanoSIMS reference literature in environmental microbiology and cell biology may be found at http://www.cameca.fr/literature/scientific-publications/NanoSIMS.aspx.

Orphan - first one to use for natrual consortuum? https://pubmed.ncbi.nlm.nih.gov/11463914/

Technical considerations

Generally, NanoSIMS uses a primary beam of ions to erode the sample surface and produce atomic collisions, some of the collisions resulting in the release of secondary ion particles. These ions are transmitted through a mass spectrometer, where the masses are measured and identified. ^[1] The primary ion beam can raster across the sample and create a ‘map’ of the element and isotope distribution by counting the number of ions that originated from each point at a 50 nanometers (nm) resolution, 10-50 times greater than convention SIMS. ^{[2] [3]} This allows for the isotopic composition of individual cells to be distinguished at ppm or ppb range.

NanoSIMS can detect minute mass differences between ions at the resolution of M/dM > 5000, where M is the nominal mass of the isotope and dM is the mass difference between the isotopes of interest. ^[4]The high mass resolution capabilities of NanoSIMS allows for different elements and their isotopes to be identified and spatially mapped in the sample, even if very close in mass. Up to 5 (NanoSIMS 50) or 7 (NanoSIMS 50 L) masses can be simultaneously detected, from hydrogen to uranium, though with limitations. ^{[1] [3]}The relatively large number of masses that can be detected per run is useful as isotope measurement errors from small changes in instrumental or sample conditions that may occur in between runs to capture a broad range of masses are avoided. ^[4] The ion beam must either be set to detect negative or positive ions, commonly completed by using a Cesium+ or Oxygen- beam, respectively. ^[5] This high mass resolution is particularly relevant to biological applications, as nitrogen is one of the most common elements in organisms. However, due to the low electron affinity of the nitrogen atom, the production of secondary ions is rare. Instead, molecules such as CN can be generated and measured. However, due to isotope combinations (such as the isobars 13C14N-, and 12C15N-), nearly identical molecular weights of 27.000 and 27.006 daltons, respectively, will be generated. Unlike other imaging techniques, NanoSIMS can safely distinguish the differences between these molecules. ^[5]

One of the most critical steps in NanoSIMS use is sample preparation. ^[6] Specific protocols should be developed for individual experiments in order to best preserve the true spatial distribution and abundance of molecules based on the sample. In general, due to the design of the NanoSIMS machine, the sample must be vacuum compatible (ie, volatile free), flat, which reduces varying ionization trajectories, and conductive, which can be accomplished sputter coating with Au, Ir, or C. Biological samples, such as cells or tissue, can be fixed and embedded in a resin before sectioning in 100 nm slices, and placed on silicon chips or slides before viewing. ^[6]

Biological Applications

Initially develops for geochemical and related fields, NanoSIMS is now utilized by a wide variety of fields, including biology and microbiology. In biomedical research, NanoSIMS is also referred to as Multi-isotope imaging mass spectrometry (MIMS). ^[7] The 50nm resolution allows unprecedented resolution of cellular and sub-cellular features (as reference, the model organic E. coli is typically 1,000 to 2,000 nm in diameter). The high mass resolution also allows for small variation in natural isotope abundance to be distinguished. ^[5]

The first use of NanoSIMS in biology was by Peteranderl and Lechene in 2004, who used a prototype of NanoSIMS to examine and measure carbon and nitrogen isotopes of eukaryotic cells. This study was the first time that carbon and nitrogen isotope ratios were directly measured at a sub-cellular scale in a biological sample. ^[8] Other microscopy techniques can be used in tandem with NanoSIMS that allow for multiple types of information (such as taxonomic information through fluorescence in situ hybridization (FISH) or identification of additional physiological features via transmission electron microscopy) to be provided. NanoSIMS can be used for pure cultures, co cultures, and mixed community samples. ^[4]

Immunogold labeling

Traditional methods that are used to label and identify subcellular features of cells, such as immunogold labeling, can also be used with NanoSIMS analysis. Immunogold labeling uses antibodies to target specific proteins, and subsequently labels the antibodies with gold nano particles. The NanoSIMS instrument can detect the gold particles, providing the location of the labelled proteins at a high scale resolution. Gold-containing or platinum-containing compounds used as anticancer drugs were imaged using NanoSIMS to examine the sub cellar distribution in breast cancer and colon cancer cells, respectively. ^[9]

Stable isotope labeling

Another common technique typically used in NanoSIMS analysis is stable isotope probing. This method involves the introduction of stable isotopically labelled biologically-relevant compounds to organisms for consumption and integration into organic matter. When analyzed via NanoSIMS, the technique is referred to as nanoSIP. ^[10] NanoSIMS can be used to detect which organisms incorporated which molecules, how much was incorporated in a semi-quantitative manner, and where in the cell the incorporation occurred. Previous quantitative analysis of stable isotopically labeled molecules involved measurement of bulk material, which did not allow for insights about the contributions of individual cells or subcellular compartments to be made. ^[11] The removal of large foreign molecules from the experimental setup alleviates concerns that tagged molecules required for other microscopy techniques may have different biochemical responses or properties than normal.

This technique can be used to study nutrient exchange. The mouse gut microbiome was investigated to determine which microbes fed on host-derived compounds. For this, mice were given food enriched in the stable isotope L-threonine and the microbial biomass examined. ^[12] NanoSIMS allows for the metabolic contributions of individual microbes to be examined. NanoSIMS was used to study and prove for the first time the nitrogen fixing abilities of bacteria and archaea from the deep ocean by supplying 15N nitrogen contain compounds to sediment samples. ^[13] NanoSIMS can also be used to estimate growth rate, as the amount of carbon or other substrate accumulated inside the cell allows for estimation of how much biomass is being generated. ^[14]

Measuring natural isotope abundance in organisms

Organic material naturally contains stable isotopes at different ratios in the environment, which can provide information on the origin of the food source for the organisms. This type of analysis was first used in 2001 in conjunction with FISH to examine syntrophic relationships between anaerobic methane-oxidizing archaea and sulfate reducing bacteria. ^[15] Isotopes with naturally low abundances may not be able to be detected with this method.

Paleobiology

NanoSIMS can also be used to examine the elemental and isotopic composition of microparticles preserved in the rock record. ^[16] The types of elements and isotopic ratios can help determine if the material is of biological origin. ^[4] NanoSIMS was first used in this field of paleobiology in 2005 by Robert et al. ^[17] In this study, microfossils were found to contain carbon, nitrogen, and sulfur elements arranged as ‘globules’ that were reminiscent of cell walls. The ratio of carbon to nitrogen measured also served as an indicator of biological origin, as the rock surrounding the fossils had very different C to N ratios. ^[16]