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 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.

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.

Ar cluster sputtering of organic multilayers such as organic light-emitting diode model structures and Irganox delta layers is studied with time-of-flight secondary ion mass spectroscopy in the dual beam mode. Results for sputtering yield volumes and depth resolution are presented for Ar clusters with sizes from 500 to 5000 atoms in the energy range from 2.5 to 20 keV. The sputtering yield volume shows a linear dependence on the energy per atom for all materials in this study with a material-dependent threshold below 1 eV/atom. The sputtering yield volume at a given energy per atom increases with the cluster size. At constant beam energies, the sputtering yield volume decreases slightly with increasing cluster size. The depth resolution is investigated for the two model systems as a function of energy and cluster size, and it will be shown that the depth resolution depends mainly on the sample roughening. The depth resolution is approximately proportional to the depth of the impact crater at a given cluster size and energy. The optimum depth resolution achieved is in the range of 4-5 nm and is fairly constant with depth. At very low energies per atom close to the threshold energy, ripple formation is observed that leads to a fast degradation of the depth resolution with depth. This can be completely eliminated by fast sample rotation. Finally, the perspective of 3D analysis of organic devices with high depth resolution in the dual beam mode will be discussed. Figure 1. a) Sputtering yield volume of HTM-1 versus energy/atom for sputtering with Ar cluster sizes from 500 to 5000. b) Sputtering yield volume of HTM-1 for Ar cluster energies from 2.5 keV to 20 keV as a function of the cluster size.

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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.

Dual beam depth profiling was applied in order to investigate the possibilities and limitations of C 60 and Ar cluster ion sputtering for depth profiling of polymer materials. Stability and intensity of characteristic high mass molecular ion signals as well as sputter yields will be compared. For this purpose, different beam energies resulting in 2-10 eV/atom for Ar n and 167-667 eV/atom for C 60 sputtering were applied to various polymer samples. From our experiments, we can conclude that most of the limitations C 60 sputtering suffers from could be successfully overcome and that the Ar gas cluster ion beam seems to be a more universal tool for sputtering of organic materials.

High resolution depth profiling has been performed in a time-of-flight secondary ion mass spectroscopy (TOF-SIMS) instrument equipped with independent ion sources for sputtering (crater formation) and for SIMS analysis. In this dual beam mode a low energy sputter gun (Cs or any gas ion) allows a free selection of optimum sputter conditions with regard to depth resolution and matrix optimization. For secondary ion generation an independent high energy ion beam, optimized with regard to focussing and secondary ion yield (Ga or gas ion source) is applied. For different sputter gases (Ar, Xe, O2, and SF6), energies (0.3–2 keV) and angles of incidence a systematic investigation of B layers in Si and GaAlAs multilayers has been carried out. Decay lengths of 0.53 nm were achieved for low energy sputtering of B layers in Si with 0.6 keV SF5+. In this dual beam mode the depth profiling performance of TOF-SIMS exceeds that of state of the art quadrupole and magnetic sector field instruments in several fields of application, important in particular in microelectronics.