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.

Argon cluster ion source evaluation on lipid standards and rat brain tissue samples.

Argon cluster ion sources for sputtering and secondary ion mass spectrometry use projectiles consisting of several hundreds of atoms, accelerated to 10-20 keV, and deposit their kinetic energy within the top few nanometers of the surface. For organic materials, the sputtering yield is high removing material to similar depth. Consequently, the exposed new surface is relatively damage free. It has thus been demonstrated on model samples that it is now really possible to perform dual beam depth profiling experiments in organic materials with this new kind of ion source. Here, this possibility has been tested directly on tissue samples, 14 µm thick rat brain sections, allowing primary ion doses much larger than the so-called static secondary ion mass spectrometry (SIMS) limit and demonstrating the possibility to enhance the sensitivity of time-of-flight (TOF)-SIMS biological imaging. However, the depth analyses have also shown some variations of the chemical composition as a function of depth, particularly for cholesterol, as well as some possible matrix effects due to the presence or absence of this compound.

Argon cluster ion beams for organic depth profiling: results from a VAMAS interlaboratory study.

The depth profiling of organic materials with argon cluster ion sputtering has recently become widely available with several manufacturers of surface analytical instrumentation producing sources suitable for surface analysis. In this work, we assess the performance of argon cluster sources in an interlaboratory study under the auspices of VAMAS (Versailles Project on Advanced Materials and Standards). The results are compared to a previous study that focused on C(60)(q+) cluster sources using similar reference materials. Four laboratories participated using time-of-flight secondary-ion mass spectrometry for analysis, three of them using argon cluster sputtering sources and one using a C(60)(+) cluster source. The samples used for the study were organic multilayer reference materials consisting of a ∼400-nm-thick Irganox 1010 matrix with ∼1 nm marker layers of Irganox 3114 at depths of ∼50, 100, 200, and 300 nm. In accordance with a previous report, argon cluster sputtering is shown to provide effectively constant sputtering yields through these reference materials. The work additionally demonstrates that molecular secondary ions may be used to monitor the depth profile and depth resolutions approaching a full width at half maximum (fwhm) of 5 nm can be achieved. The participants employed energies of 2.5 and 5 keV for the argon clusters, and both the sputtering yields and depth resolutions are similar to those extrapolated from C(60)(+) cluster sputtering data. In contrast to C(60)(+) cluster sputtering, however, a negligible variation in sputtering yield with depth was observed and the repeatability of the sputtering yields obtained by two participants was better than 1%. We observe that, with argon cluster sputtering, the position of the marker layers may change by up to 3 nm, depending on which secondary ion is used to monitor the material in these layers, which is an effect not previously visible with C(60)(+) cluster sputtering. We also note that electron irradiation, used for charge compensation, can induce molecular damage to areas of the reference samples well beyond the analyzed region that significantly affects molecular secondary-ion intensities in the initial stages of a depth profile in these materials.

Imaging G-SIMS: a novel bismuth-manganese source emitter.

G-SIMS (gentle-SIMS) is a powerful method that considerably simplifies complex static secondary ion mass spectrometry (SSIMS) analysis of organics at surfaces. G-SIMS uses two primary ion beams that generate high and low fragmentation conditions at the surface. This allows an extrapolation to equivalent experimental conditions with very low fragmentation. Consequently, the spectra are less complex, contain more structural information and are simpler to interpret. In general, G-SIMS spectra more closely resemble electron ionisation mass spectra than SSIMS spectra. A barrier for the wider uptake of G-SIMS is the requirement for two ion beams producing suitably different fragmentation conditions and the need for their spatial registration (spatial alignment) at the surface, which is especially important for heterogeneous samples. The most popular source is the liquid metal ion source (LMIS), which is now sold with almost every new time-of-flight (TOF)-SIMS instrument. Here, we have developed a novel bismuth-manganese emitter (the 'G-tip') for the popular LMISs. This simplifies the alignment and gives excellent G-SIMS imaging and spectroscopy without significantly compromising the bismuth cluster ion beam performance.

Improvement of biological time-of-flight-secondary ion mass spectrometry imaging with a bismuth cluster ion source.

A new liquid metal ion gun (LMIG) filled with bismuth has been fitted to a time-of-flight-secondary ion mass spectrometer (TOF-SIMS). This source provides beams of Bi(n)q+ clusters with n = 1-7 and q = 1 and 2. The appropriate clusters have much better intensities and efficiencies than the Au3+ gold clusters recently used in TOF-SIMS imaging, and allow better lateral and mass resolution. The different beams delivered by this ion source have been tested for biological imaging of rat brain sections. The results show a great improvement of the imaging capabilities in terms of accessible mass range and useful lateral resolution. Secondary ion yields Y, disappearance cross sections sigma, efficiencies E = Y/sigma , and useful lateral resolutions deltaL have been compared using the different bismuth clusters, directly onto the surface of rat brain sections and for several positive and negative secondary ions with m/z ranging from 23 up to more than 750. The efficiency and the imaging capabilities of the different primary ions are compared by taking into account the primary ion current for reasonable acquisition times. The two best primary ions are Bi3+ and Bi5(2+). The Bi3+ ion beam has a current at least five times larger than Au3+ and therefore is an excellent beam for large-area imaging. Bi5(2+) ions exhibit large secondary ions yields and a reasonable intensity making them suitable for small-area images with an excellent sensitivity and a possible useful lateral resolution <400 nm.