Melissa K. Passarelli

Lipid Imaging with Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS)

Fundamental advances in secondary ion mass spectrometry (SIMS) now allow for the examination and characterization of lipids directly from biological materials. The successful application of SIMS-based imaging in the investigation of lipids directly from tissue and cells are demonstrated. Common complications and technical pitfalls are discussed. In this review, we examine the use of cluster ion sources and cryogenically compatible sample handling for improved ion yields and to expand the application potential of SIMS. Methodological improvements, including pre-treating the sample to improve ion yields and protocol development for 3-dimensional analyses (i.e. molecular depth profiling), are also included in this discussion. New high performance SIMS instruments showcasing the most advanced instrumental developments, including tandem MS capabilities and continuous ion beam compatibility, are described and the future direction for SIMS in lipid imaging is evaluated.

C60 secondary ion mass spectrometry with a hybrid-quadrupole orthogonal time-of-flight mass spectrometer.

A hybrid quadrupole orthogonal time-of-flight mass spectrometer optimized for matrix-assisted laser desorption ionization (MALDI) and electrospray ionization has been equipped with a C 60 cluster ion source. This configuration is shown to exhibit a number of characteristics that improve the performance of traditional time-of-flight secondary ion mass spectrometry (TOF-SIMS) experiments for the analysis of complex organic materials and, potentially, for chemical imaging. Specifically, the primary ion beam is operated as a continuous rather than a pulsed beam, resulting in up to 4 orders of magnitude greater ion fluence on the target. The secondary ions are extracted at very low voltage into 8 mTorr of N 2 gas introduced for collisional focusing and cooling purposes. This extraction configuration is shown to yield secondary ions that rapidly lose memory of the mechanism of their birth, yielding tandem mass spectra that are identical for SIMS and MALDI. With implementation of ion trapping, the extraction efficiency is shown to be equivalent to that found in traditional TOF-SIMS machines. Examples are given, for a variety of substrates that illustrate mass resolution of 12,000-15,600 with a mass range for inorganic compounds to m/ z 40,000. Preliminary chemical mapping experiments show that with added sensitivity, imaging in the MS/MS mode of operation is straightforward. In general, the combination of MALDI and SIMS is shown to add capabilities to each technique, providing a robust platform for TOF-SIMS experiments that already exists in a large number of laboratories.

Single-Cell Analysis: Visualizing Pharmaceutical and Metabolite Uptake in Cells with Label-Free 3D Mass Spectrometry Imaging.

Detecting metabolites and parent compound within a cell type is now a priority for pharmaceutical development. In this context, three-dimensional secondary ion mass spectrometry (SIMS) imaging was used to investigate the cellular uptake of the antiarrhythmic agent amiodarone, a phospholipidosis-inducing pharmaceutical compound. The high lateral resolution and 3D imaging capabilities of SIMS combined with the multiplex capabilities of ToF mass spectrometric detection allows for the visualization of pharmaceutical compound and metabolites in single cells. The intact, unlabeled drug compound was successfully detected at therapeutic dosages in macrophages (cell line: NR8383). Chemical information from endogenous biomolecules was used to correlate drug distributions with morphological features. From this spatial analysis, amiodarone was detected throughout the cell, with the majority of the compound found in the membrane and subsurface regions and absent in the nuclear regions. Similar results were obtained when the macrophages were doped with amiodarone metabolite, desethylamiodarone. The fwhm lateral resolution measured across an intracellular interface in high lateral resolution ion images was approximately 550 nm. Overall, this approach provides the basis for studying cellular uptake of pharmaceutical compounds and their metabolites on the single cell level.

Single-Cell Lipidomics: Characterizing and Imaging Lipids on the Surface of Individual Aplysia californica Neurons with Cluster Secondary Ion Mass Spectrometry

Neurons isolated from Aplysia californica , an organism with a well-defined neural network, were imaged with secondary ion mass spectrometry, C(60)-SIMS. A major lipid component of the neuronal membrane was identified as 1-hexadecyl-2-octadecenoyl-sn-glycero-3-phosphocholine [PC(16:0e/18:1)] using tandem mass spectrometry (MS/MS). The assignment was made directly off the sample surface using a C(60)-QSTAR instrument, a prototype instrument that combines an ion source with a commercial electrospray ionization/matrix-assisted laser desorption ionization (ESI/MALDI) mass spectrometer. Normal phase liquid chromatography mass spectrometry (NP-LC-MS) was used to confirm the assignment. Cholesterol and vitamin E were also identified with in situ tandem MS analyses that were compared to reference spectra obtained from purified compounds. In order to improve sensitivity on the single-cell level, the tandem MS spectrum of vitamin E reference material was used to extract and compile all the vitamin E related peaks from the cell image. The mass spectrometry images reveal heterogeneous distributions of intact lipid species, PC(16:0e/18:1), vitamin E, and cholesterol on the surface of a single neuron. The ability to detect these molecules and determine their relative distribution on the single-cell level shows that the C(60)-QSTAR is a potential platform for studying important biochemical processes, such as neuron degeneration.

Single-Cell Imaging Mass Spectrometry

Single-cell imaging mass spectrometry (IMS) is a powerful technique used to map the distributions of endogenous biomolecules with subcellular resolution. Currently, secondary ion mass spectrometry is the predominant technique for single-cell IMS, thanks to its submicron lateral resolution and surface sensitivity. However, recent methodological and technological developments aimed at improving the spatial resolution of matrix assisted laser desorption ionization (MALDI) have made this technique a potential platform of single-cell IMS. MALDI opens the field of single-cell IMS to new possibilities, including single cell proteomic imaging and atmospheric pressure analyses; however, sensitivity is a challenge. In this report, we estimate the availability of proteins and lipids in a single cell and discuss strategies employed to improve sensitivity at the single-cell level.

Chemical intervention in plant sugar signalling increases yield and resilience

The pressing global issue of food insecurity due to population growth, diminishing land and variable climate can only be addressed in agriculture by improving both maximum crop yield potential and resilience. Genetic modification is one potential solution, but has yet to achieve worldwide acceptance, particularly for crops such as wheat. Trehalose-6-phosphate (T6P), a central sugar signal in plants, regulates sucrose use and allocation, underpinning crop growth and development. Here we show that application of a chemical intervention strategy directly modulates T6P levels in planta. Plant-permeable analogues of T6P were designed and constructed based on a ‘signalling-precursor’ concept for permeability, ready uptake and sunlight-triggered release of T6P in planta. We show that chemical intervention in a potent sugar signal increases grain yield, whereas application to vegetative tissue improves recovery and resurrection from drought. This technology offers a means to combine increases in yield with crop stress resilience. Given the generality of the T6P pathway in plants and other small-molecule signals in biology, these studies suggest that suitable synthetic exogenous small-molecule signal precursors can be used to directly enhance plant performance and perhaps other organism function.

Development of an Organic Lateral Resolution Test Device for Imaging Mass Spectrometry

An organic lateral resolution test device has been developed to measure the performance of imaging mass spectrometry (IMS) systems. The device contains periodic gratings of polyethylene glycol (PEG) and lipid bars covering a wide range of spatial frequencies. Microfabrication technologies were employed to produce well-defined chemical interfaces, which allow lateral resolution to be assessed using the edge-spread function (ESF). In addition, the design of the device allows for the direct measurement of the modulation transfer function (MTF) to assess image quality. Scanning electron microscopy (SEM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) were used to characterize the device. TOF-SIMS imaging was used to measure the chemical displacement of biomolecules in matrix-assisted laser desorption/ionization (MALDI) matrix crystals. In a proof-of-concept experiment, the platform was also used to evaluate MALDI matrix application methods, specifically aerosol spray and sublimation methods.

The 3D OrbiSIMS—label-free metabolic imaging with subcellular lateral resolution and high mass-resolving power

We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 μm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmitters—gamma-aminobutyric acid, dopamine and serotonin—with high spectroscopic confidence in the mouse hippocampus. We also putatively annotated and mapped the subcellular localization of 29 sulfoglycosphingolipids and 45 glycerophospholipids, and we confirmed lipid identities with tandem mass spectrometry. We demonstrated single-cell metabolomic profiling using rat alveolar macrophage cells incubated with different concentrations of the drug amiodarone, and we observed that the upregulation of phospholipid species and cholesterol is correlated with the accumulation of amiodarone.

A Novel Hybrid Dual Analyzer SIMS Instrument for Improved Surface and 3D-Analysis

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an established, highly sensitive analytical technique for mass spectrometry (MS) imaging applications with a lateral resolution below 100 nm. Elemental and molecular information is obtained by bombarding the surface with a focused primary ion beam and analyzing the generated secondary ions in a TOF mass analyzer. Furthermore 3D imaging is possible by employing a lower energetic quasi DC sputter beam for material removal (sputter cycle) and a short pulsed small spot analysis beam for optimal mass spectral and imaging performance (so-called dual beam mode). Application of this technique for the localization of drugs and their metabolites in drug-doped cells could be used to find regions in which a pharmaceutical compound accumulates. This would be extremely helpful for selection of possible drug candidates in pre-clinical studies, thereby reducing the development costs for new pharmaceutical products. Furthermore surveying biologically relevant molecules, like lipids, in tissue can give valuable information on the molecular fundamentals of diseases and the effects of treatments.

Intracellular Drug Uptake—A Comparison of Single Cell Measurements Using ToF-SIMS Imaging and Quantification from Cell Populations with LC/MS/MS

ToF-SIMS is a label-free imaging method that has been shown to enable imaging of amiodarone in single rat macrophage (NR8383) cells. In this study, we show that the method extends to three other cell lines relevant to drug discovery: human embryonic kidney (HEK293), cervical cancer (HeLa), and liver cancer (HepG2). There is significant interest in the variation of drug uptake at the single cell level, and we use ToF-SIMS to show that there is great diversity between individual cells and when comparing each of the cell types. These single cell measurements are compared to quantitative measurements of cell-associated amiodarone for the population using LC/MS/MS and cell counting with flow cytometry. NR8383 and HepG2 cells uptake the greatest amount of amiodarone with an average of 2.38 and 2.60 pg per cell, respectively, and HeLa and Hek 293 have a significantly lower amount of amiodarone at 0.43 and 0.36 pg per cell, respectively. The amount of cell-associated drug for the ensemble population measurement (LC/MS/MS) is compared with the ToF-SIMS single cell data: a similar amount of drug was detected per cell for the NR8383, and HepG2 cells at a greater level than that for the HEK293 cells. However, the two techniques did not agree for the HeLa cells, and we postulate potential reasons for this.