Molecular Depth Profiling with Argon Gas Cluster Ion Beams

Argon gas cluster ion beams (Ar-GCIBs) are remarkable new projectiles for secondary ion mass spectrometry (SIMS) depth profiling of organic materials. However, the optimal cluster size and kinetic energy to provide the best quality of depth profiles, in terms of high ionization efficiency of the target molecules, little chemical damage, and short experiment time, for organic materials is not fully understood. Hence, the effect of cluster size and kinetic energy on the quality of molecular depth profiling is investigated on a simple platform composed of trehalose thin films to acquire more fundamental information about the ion/solid interaction. The results suggest that the sputter yield (<i>Y</i>/<i>n</i>) of argon clusters is linearly dependent upon kinetic energy per atom (<i>E</i>/<i>n</i>). When <i>E</i>/<i>n</i> > 5 eV/atom, normal depth profiles are obtained with relatively high sputter yields. When <i>E</i>/<i>n</i> ≤ 5 eV/atom, however, distorted depth profiles in the steady state region are observed, which exhibit a low sputter yield and variable ionization efficiency. As a consequence of these observations, it was concluded that high kinetic energy increases the useful molecular ion yield of trehalose and that Ar_<i>n</i>⁺ clusters with a small <i>E</i>/<i>n</i> value minimize ion beam bombardment induced chemical damage. Hence optimal conditions for molecular depth profiling will be obtained using the highest kinetic energy with the largest clusters while maintaining a value of <i>E</i>/<i>n</i> near a threshold value of 5 eV/atom. In general, this study provides insight into selecting optimal Ar-GCIB characteristics for molecular depth profiling of organic materials

Strong Field Ionization of β‑Estradiol in the IR: Strategies To Optimize Molecular Postionization in Secondary Neutral Mass Spectrometry

Strong field laser photoionization of β-estradiol in the 10¹³–10¹⁵ W/cm² intensity range at wavelengths between 1200 and 2000 nm was examined for both evaporated gas phase molecules and neutral species sputtered from a solid surface under bombardment with 20 keV C₆₀⁺ ions. It is shown that the ionization efficiency is saturated at laser intensities of the order of 10¹³ W/cm², while laser-induced photofragmentation is found to strongly increase between 10¹³ and 10¹⁴ W/cm² and stay constant at higher intensities. These findings suggest a strategy to improve the postionization efficiency by defocusing the laser beam in order to sample a larger fraction of the sputtered plume, while at the same time optimizing the conditions for intact photoionization of the sputtered molecules. The results reveal a substantial enhancement of the signal of intact postionized molecular ions to a level an order of magnitude above that of the corresponding secondary ions. They also provide an experimental estimate of ∼2.5 × 10^–2 as an upper bound for the ion fraction (secondary ion formation probability) of the sputtered molecules

Reducing the Matrix Effect in Molecular Secondary Ion Mass Spectrometry by Laser Post-Ionization

Strategies to reduce and overcome matrix effects in molecular secondary ion mass spectrometry (SIMS) are investigated using laser-based post-ionization of sputtered neutral organic molecules released under C₆₀⁺ bombardment. Using a two-component multilayer film similar to that employed in a recent VAMAS interlaboratory study, SIMS depth profiles of the protonated and deprotonated quasi-molecular ions of two well-studied organic molecules, Irganox 1010 and Irganox 1098, were measured along with that of the corresponding neutral precursor molecules. When compared to composition-dependent ionization probability changes of the secondary ions, the resulting profiles are much improved. We demonstrate that detection of neutral molecules via laser post-ionization yields significantly reduced matrix effects when compared to SIMS depth profiles in both positive and negative secondary ion mode. These results suggest that this approach may provide a useful pathway for acquiring depth profiles from complex organic samples with improved capabilities for quantitation

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, C₆₀-SIMS. A major lipid component of the neuronal membrane was identified as 1-hexadecyl-2-octadecenoyl-<i>sn</i>-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 C₆₀-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 C₆₀-QSTAR is a potential platform for studying important biochemical processes, such as neuron degeneration

Subcellular Chemical Imaging of Antibiotics in Single Bacteria Using C₆₀-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. The methodology was further validated by the observation of reduction in tetracycline accumulation in an E. coli strain expressing the tetracycline-specific efflux pump (TetA) compared to the isogenic control. This study serves as a proof-of-concept for a new strategy for chemical imaging at the nanoscale and has the potential to aid discovery of new antibacterials

Depth Profiling of Metal Overlayers on Organic Substrates with Cluster SIMS

Molecular depth profiling of organic thin films by erosion with energetic cluster ion beams is a unique aspect of secondary ion mass spectrometry (SIMS) experiments. Although depth profiles of complex multilayer organic structures can be acquired with little damage accumulation and with depth resolution of <10 nm using either C₆₀⁺ or Ar_<i>x</i>⁺ with <i>x</i> = 500–5000, hybrid materials consisting of both organic and inorganic layers often yield poor results. To unravel the factors that lead to this difficulty, we developed a model system composed of a thin gold layer of 1.4 to 3.5 nm deposited either on top of or sandwiched within a cholesterol thin film matrix which is several hundred nanometers thick. For these systems, the results show that by erosion with a 40 keV C₆₀⁺ beam, reliable depth profiles can always be acquired as indicated by the presence of a steady state molecular ion signal. During the erosion process, however, gold atoms from the gold overlayer are implanted into the cholesterol matrix beneath it, resulting in a reduced sputter yield, an increase in the amount of cholesterol fragmentation and an increase in the thickness of the cluster ion-induced altered layer. The results also show that the effects of the metal film on the organic substrate are independent of the gold film thickness once the film thickness exceeds 1.4 nm. In general, this model study provides mechanistic insight into the depth profiling of heterogeneous thin film structures and offers a possible path for improving the quality of the depth profiles by employing low energy atomic ion sputtering in the region of the metal layer

Near Infrared (NIR) Strong Field Ionization and Imaging of C₆₀ Sputtered Molecules: Overcoming Matrix Effects and Improving Sensitivity

Strong field ionization (SFI) was applied for the secondary neutral mass spectrometry (SNMS) of patterned rubrene films, mouse brain sections, and Botryococcus braunii algal cell colonies. Molecular ions of rubrene, cholesterol, C₃₁ diene/triene, and three wax monoesters were detected, representing some of the largest organic molecules ever ionized intact by a laser post-ionization experiment. In rubrene, the SFI SNMS molecular ion signal was ∼4 times higher than in the corresponding secondary-ion mass spectroscopy (SIMS) analysis. In the biological samples, the achieved signal improvements varied among molecules and sampling locations, with SFI SNMS, in some cases, revealing analytes made completely undetectable by the influence of matrix effects in SIMS

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. Participants were asked to estimate the volume fractions in two of the layers and were provided with the compositions of all other layers. Participants using XPS provided volume fractions within 0.03 of the nominal values. Participants using ToF-SIMS either made no attempt, or used various methods that gave results ranging in error from 0.02 to over 0.10 in volume fraction, the latter representing a 50% relative error for a nominal volume fraction of 0.2. Error was predominantly caused by inadequacy in the ability to compensate for primary ion intensity variations and the matrix effect in SIMS. Matrix effects in these materials appear to be more pronounced as the number of atoms in both the primary analytical ion and the secondary ion increase. Using the participants’ data we show that organic SIMS matrix effects can be measured and are remarkably consistent between instruments. We provide recommendations for identifying and compensating for matrix effects. Finally, we demonstrate, using a simple normalization method, that virtually all ToF-SIMS participants could have obtained estimates of volume fraction that were at least as accurate and consistent as XPS