Multimodal mass spectrometry imaging identifies cell-type-specific metabolic and lipidomic variation in the mammalian liver.

Spatial single-cell omics provides a readout of biochemical processes. It is challenging to capture the transient lipidome/metabolome from cells in a native tissue environment. We employed water gas cluster ion beam secondary ion mass spectrometry imaging ([HO]-GCIB-SIMS) at ≤3 μm resolution using a cryogenic imaging workflow.

Successive High-Resolution (HO)-GCIB and C-SIMS Imaging Integrates Multi-Omics in Different Cell Types in Breast Cancer Tissue.

The temporo-spatial organization of different cells in the tumor microenvironment (TME) is the key to understanding their complex communication networks and the immune landscape that exists within compromised tissues. Multi-omics profiling of single-interacting cells in the native TME is critical for providing further information regarding the reprograming mechanisms leading to immunosuppression and tumor progression. This requires new technologies for biomolecular profiling of phenotypically heterogeneous cells on the same tissue sample.

Multiomics Imaging Using High-Energy Water Gas Cluster Ion Beam Secondary Ion Mass Spectrometry [(HO)-GCIB-SIMS] of Frozen-Hydrated Cells and Tissue.

Integration of multiomics at the single-cell level allows the unambiguous dissecting of phenotypic heterogeneity at different states such as health, disease, and biomedical response. Imaging mass spectrometry holds the promise of being able to measure multiple types of biomolecules in parallel in the same cell. We have explored the possibility of using water gas cluster ion beam secondary ion mass spectrometry [(HO)-GCIB-SIMS] as an analytical tool for multiomics assay.

Direct Mapping of Phospholipid Ferroptotic Death Signals in Cells and Tissues by Gas Cluster Ion Beam Secondary Ion Mass Spectrometry (GCIB-SIMS).

Peroxidized phosphatidylethanolamine (PEox) species have been identified by liquid chromatography mass spectrometry (LC-MS) as predictive biomarkers of ferroptosis, a new program of regulated cell death. However, the presence and subcellular distribution of PEox in specific cell types and tissues have not been directly detected by imaging protocols. By applying gas cluster ion beam secondary ion mass spectrometry (GCIB-SIMS) imaging with a 70 keV (H O) (n>28 000) cluster ion beam, we were able to map PEox with 1.

Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells.

Metabolons, multiprotein complexes consisting of sequential enzymes of a metabolic pathway, are proposed to be biosynthetic "hotspots" within the cell. However, experimental demonstration of their presence and functions has remained challenging. We used metabolomics and in situ three-dimensional submicrometer chemical imaging of single cells by gas cluster ion beam secondary ion mass spectrometry (GCIB-SIMS) to directly visualize de novo purine biosynthesis by a multienzyme complex, the purinosome.

Enhanced Ion Yields Using High Energy Water Cluster Beams for Secondary Ion Mass Spectrometry Analysis and Imaging.

Previous studies have shown that the use of a 20 keV water cluster beam as a primary beam for the analysis of organic and bio-organic systems resulted in a 10-100 times increase in positive molecular ion yield for a range of typical analytes compared to C and argon cluster beams. This resulted in increased sensitivity to important lipid molecules in the bioimaging of rat brain. Building on these studies, the present work compares 40 and 70 keV water cluster beams with cluster beams composed of pure argon, argon and 10%CO, and pure CO.

Secondary-Ion Mass Spectrometry Images Cardiolipins and Phosphatidylethanolamines at the Subcellular Level.

Millions of diverse molecules constituting the lipidome act as important signals within cells. Of these, cardiolipin (CL) and phosphatidylethanolamine (PE) participate in apoptosis and ferroptosis, respectively. Their subcellular distribution is largely unknown.

C-O Bond Dissociation and Induced Chemical Ionization Using High Energy (CO) Gas Cluster Ion Beam.

A gas cluster ion beam (GCIB) source, consisting of CO clusters and operating with kinetic energies of up to 60 keV, has been developed for the high resolution and high sensitivity imaging of intact biomolecules. The CO molecule is an excellent molecule to employ in a GCIB source due to its relative stability and improved focusing capabilities, especially when compared to the conventionally employed Ar cluster source. Here we report on experiments aimed to examine the behavior of CO clusters as they impact a surface under a variety of conditions.

Gas Cluster Ion Beams for Secondary Ion Mass Spectrometry.

Gas cluster ion beams (GCIBs) provide new opportunities for bioimaging and molecular depth profiling with secondary ion mass spectrometry (SIMS). These beams, consisting of clusters containing thousands of particles, initiate desorption of target molecules with high yield and minimal fragmentation. This review emphasizes the unique opportunities for implementing these sources, especially for bioimaging applications.

Subcellular Chemical Imaging of Antibiotics in Single Bacteria Using C-Secondary Ion Mass Spectrometry.

The inherent difficulty of discovering new and effective antibacterials and the rapid development of resistance particularly in Gram-negative bacteria, illustrates the urgent need for new methods that enable rational drug design. Here we report the development of 3D imaging cluster Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) as a label-free approach to chemically map small molecules in aggregated and single Escherichia coli cells, with ∼300 nm spatial resolution and high chemical sensitivity. The feasibility of quantitative analysis was explored, and a nonlinear relationship between treatment dose and signal for tetracycline and ampicillin, two clinically used antibacterials, was observed.

Gas Cluster Ion Beam Time-of-Flight Secondary Ion Mass Spectrometry High-Resolution Imaging of Cardiolipin Speciation in the Brain: Identification of Molecular Losses after Traumatic Injury.

Gas cluster ion beam-secondary ion mass spectrometry (GCIB-SIMS) has shown the full potential of mapping intact lipids in biological systems with better than 10 μm lateral resolution. This study investigated further the capability of GCIB-SIMS in imaging high-mass signals from intact cardiolipin (CL) and gangliosides in normal brain and the effect of a controlled cortical impact model (CCI) of traumatic brain injury (TBI) on their distribution. A combination of enzymatic and chemical treatments was employed to suppress the signals from the most abundant phospholipids (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) and enhance the signals from the low-abundance CLs and gangliosides to allow their GCIB-SIMS detection at 8 and 16 μm spatial resolution.

Label-free visualization of nilotinib-functionalized gold nanoparticles within single mammalian cells by C- SIMS imaging.

Obtaining a comprehensive grasp of the behavior and interaction of pharmaceutical compounds within single cells provides some of the fundamental details necessary for more effective drug development. In particular, the changes ensuing in the carrier, drug, and host environment in targeted drug therapy applications must be explored in greater detail, as these are still not well understood. Here, nilotinib-functionalized gold nanoparticles are examined within single mammalian cells with use of imaging cluster secondary ion mass spectrometry in a model study designed to enhance our understanding of what occurs to these particles once that have been internalized.

On the SIMS Ionization Probability of Organic Molecules.

The prospect of improved secondary ion yields for secondary ion mass spectrometry (SIMS) experiments drives innovation of new primary ion sources, instrumentation, and post-ionization techniques. The largest factor affecting secondary ion efficiency is believed to be the poor ionization probability (α) of sputtered material, a value rarely measured directly, but estimated to be in some cases as low as 10. Our lab has developed a method for the direct determination of α in a SIMS experiment using laser post-ionization (LPI) to detect neutral molecular species in the sputtered plume for an organic compound.

Reducing the Matrix Effect in Organic Cluster SIMS Using Dynamic Reactive Ionization.

Dynamic reactive ionization (DRI) utilizes a reactive molecule, HCl, which is doped into an Ar cluster projectile and activated to produce protons at the bombardment site on the cold sample surface with the presence of water. The methodology has been shown to enhance the ionization of protonated molecular ions and to reduce salt suppression in complex biomatrices. In this study, we further examine the possibility of obtaining improved quantitation with DRI during depth profiling of thin films.

Reduce the matrix effect in biological tissue imaging using dynamic reactive ionization and gas cluster ion beams.

In the context of a secondary ion mass spectrometry (SIMS) experiment, dynamic reactive ionization (DRI) involves introducing a reactive dopant, HCl, into an Ar gas cluster primary ion beam along with a source of water to enable dissociation of HCl to free protons. This concerted effect, precisely occurring at the impact site of the cluster beam, enhances the protonation of molecular species. Here, the authors apply this methodology to study the hippocampus and cerebellum region of a frozen-hydrated mouse brain section.

Multimodal image fusion with SIMS: Preprocessing with image registration.

In order to utilize complementary imaging techniques to supply higher resolution data for fusion with secondary ion mass spectrometry (SIMS) chemical images, there are a number of aspects that, if not given proper consideration, could produce results which are easy to misinterpret. One of the most critical aspects is that the two input images must be of the same exact analysis area. With the desire to explore new higher resolution data sources that exists outside of the mass spectrometer, this requirement becomes even more important.

Cluster secondary ion mass spectrometry imaging of interfacial reactions of TiO2 microspheres embedded in ionic liquids.

Rationale: Our goal is to develop protocols for the elucidation of the identity and structure of reaction products embedded in a reaction medium. Results should find significance in a variety of disciplines ranging from the study of biological cells and tissues, to the steps associated with the functionalization of nanoparticles.

Methods: We utilize cluster secondary ion mass spectrometry (cluster-SIMS) to acquire three-dimensional (3D) information about 5-30 µm TiO2 microspheres imbedded into an ionic liquid.

C60-SIMS imaging of nanoparticles within mammalian cells.

To achieve successful drug delivery via nanoparticles the interactions between the nanoparticle and the chemistry of the surrounding biological environment is of central importance. A thorough understanding of these interactions is necessary in order to better elucidate information regarding drug pathways and mechanisms of action in treatment protocols. As such, it is important to identify the location of the nanoparticle, the state of its functionalization, as well as any changes in the cellular environment.

Investigations Into the Interactions of a MALDI Matrix with Organic Thin Films Using C SIMS Depth Profiling.

Molecular depth profiling of multilayer organic films is now an established protocol for cluster secondary ion mass spectrometry (SIMS). This unique capability is exploited here to study the ionization mechanism associated with matrix-enhanced SIMS and possibly matrix assisted laser desorption/ionization (MALDI). Successful depth profiling experiments were performed on model bi-layer systems using 2,5-dihydroxybenzoic acid (DHB) as the matrix with dipalmitoylphosphatidylcholine (DPPC) or phenylalanine (PHE).

Dynamic Reactive Ionization with Cluster Secondary Ion Mass Spectrometry.

Gas cluster ion beams (GCIB) have been tuned to enhance secondary ion yields by doping small gas molecules such as CH4, CO2, and O2 into an Ar cluster projectile, Arn  + (n = 1000–10,000) to form a mixed cluster. The ‘tailored beam’ has the potential to expand the application of secondary ion mass spectrometry for two- and three-dimensional molecular specific imaging. Here, we examine the possibility of further enhancing the ionization by doping HCl into the Ar cluster.

Molecular imaging of biological tissue using gas cluster ions.

An Ar ( = 1-6000) gas cluster ion source has been utilized to map the chemical distribution of lipids in a mouse brain tissue section. We also show that the signal from high mass species can be further enhanced by doping a small amount of CH into the Ar cluster to enhance the ionization of several biologically important molecules. Coupled with secondary ion mass spectrometry instrumentation which utilizes a continuous Ar cluster ion projectile, maximum spatial resolution and maximum mass resolution can be achieved at the same time.

Measuring Compositions in Organic Depth Profiling: Results from a VAMAS Interlaboratory Study.

We report the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study on the measurement of composition in organic depth profiling. Layered samples with known binary compositions of Irganox 1010 and either Irganox 1098 or Fmoc-pentafluoro-l-phenylalanine in each layer were manufactured in a single batch and distributed to more than 20 participating laboratories. The samples were analyzed using argon cluster ion sputtering and either X-ray photoelectron spectroscopy (XPS) or time-of-flight secondary ion mass spectrometry (ToF-SIMS) to generate depth profiles.