Matrix-Enhanced SIMS: The Influence of Primary Ion Species and Cluster Size on Ion Yield and Ion Yield Enhancement of Lipids.

Time-of-flight secondary ion mass spectrometry is one of the most promising techniques for label-free analysis of biomolecules with nanoscale spatial resolution. However, high-resolution imaging of larger biomolecules such as phospholipids and peptides is often hampered by low yields of molecular ions. Matrix-enhanced SIMS (ME-SIMS), in which an organic matrix is added to the sample, is one promising approach to enhancing the ion yield for biomolecules. Optimizing this approach has, however, been challenging because the processes involved in increasing the ion yield in ME-SIMS are not yet fully understood. In this work, the matrix α-cyano-4-hydroxycinnamic acid (HCCA) has been combined with cluster primary ion analysis to better understand the roles of proton donation and reduced fragmentation on lipid molecule ion yield. A model system consisting of 1:100 mol ratio dipalmitoylphosphatidylcholine (DPPC) in HCCA as well as an HCCA-coated mouse brain cryosection have been studied using a range of Bi and Ar cluster ions. Although the molecular ion yield increased with an increase in cluster ion size, the enhancement of the signals from intact lipid molecules decreased with an increase in cluster ion size for both the model system and the mouse brain. Additionally, in both systems, protonated molecular ions were significantly more enhanced than sodium and potassium cationized molecules for all of the primary ions utilized. For the model system, the DPPC molecular ion yield was increased by more than an order of magnitude for all of the primary ions studied, and fragmentation of DPPC was dramatically reduced. However, on the brain sample, even though the HCCA matrix reduced DPPC fragmentation for all of the primary ions studied, the matrix coating suppressed the ion yield for some lipids when the larger cluster primary ions were employed. This indicated insufficient migration of the lipids into the matrix coating, so that dilution by the matrix overpowered the enhancement effect. This study provides strong evidence that the HCCA matrix both enhances protonation and reduces fragmentation. For imaging applications, the ability of the analytes to migrate to the surface of the matrix coating is also a critical factor for useful signal enhancement. This work demonstrates that the HCCA matrix provides a softer desorption environment when using Bi cluster ions than that obtained using the large gas cluster ions studied alone, indicating the potential for improved high spatial resolution imaging with ME-SIMS.

3D Molecular ToF-SIMS Imaging of Artificial Lipid Membranes Using a Discriminant Analysis-Based Algorithm.

Artificial lipid membranes play a growing role in technical applications such as biosensors in pharmacological research and as model systems in the investigation of biological lipid films. In the standard procedure for displaying the distribution of membrane components, fluorescence microscopy, the fluorophores used can influence the distribution of the components and usually not all substances can be displayed at the same time. The discriminant analysis-based algorithm used in combination with scanning time-of-flight secondary ion mass spectrometry (ToF-SIMS) enables marker-free, quantitative, simultaneous recording of all membrane components. These data are used for reconstruction of distribution patterns. In the model system used for this survey, a tear fluid lipid layer, the distribution patterns of all lipids correlate well in calculated ToF-SIMS images and epi-fluorescence microscopic images. All epi-fluorescence microscopically viewable structures are visible when using both positive and negative secondary ions and can be reproduced with high lateral resolution in the submicrometer range despite the very low signal intensity and a very low signal-to-noise ratio. In addition, three-dimensional images can be obtained with a subnanometer depth resolution. Furthermore, structures and the distribution of substances that cannot be made visible by epi-fluorescence microscopy can be displayed. This enables new insights that cannot be gained by epi-fluorescence microscopy alone.

Laser postionization of neutral molecules sputtered using bismuth and argon cluster primary ions.

In this study, the influence of two different cluster primary ions in laser secondary neutral mass spectrometry (Laser-SNMS) has been investigated. Despite the many advantages of Laser-SNMS, fragmentation of neutral organic molecules during both sputtering and photoionization has limited its efficiency for the study of large organic and biological molecules. Cluster ion sputtering, and in particular large argon gas cluster sputtering, has been proposed as a means of reducing this fragmentation. Molecules of 9-fluorenylmethoxycarbonyl-pentafluoro-l-phenylalanine were sputtered using Bi3+ and Ar2000+ cluster primary ions, and the desorbed neutral species ("secondary neutrals") were postionized using a 7.87 eV vacuum ultraviolet laser light fluorine excimer laser. By varying timing parameters and laser power density, time-of-flight and laser power density distributions were obtained to investigate the fragmentation and energy distributions of the sputtered neutral species. Changing from 30 keV Bi3+ sputtering to 10 keV Ar2000+ resulted in a significant reduction in fragmentation of the molecule as well as a suppression of the high background that results from metastable decay of highly excited ions, yielding significantly improved detection of the intact molecule and characteristic fragments. Analysis of the influence of laser power density and laser pulse delay time indicates a reduction of fragmentation in both the sputtering phase and the photoionization phase. This study demonstrates the importance of soft desorption for efficient laser postionization of large organic molecules and shows the potential for improving the efficiency of laser postionization by using large gas cluster ion sputtering.

Development of a Knudsen-type matrix coater for sample preparation for mass spectrometry imaging.

The use of time-of-flight secondary ion mass spectrometry (SIMS) is of increasing interest for biological and medical applications due to its ability to provide chemical information on a submicrometer scale. However, the detection of larger biomolecules such as phospholipids and peptides is often inhibited by high fragmentation rates and low ionization efficiencies. One way to increase the secondary ion molecular yield is to chemically modify the surface using the matrix-enhanced SIMS approach, where an organic matrix is placed upon the surface. In this study, a Knudsen cell type matrix coater was developed in order to produce well-defined thicknesses of a matrix on a sample in order to study the effect of these matrix layers on the secondary ions. Using this technique, an order of magnitude enhancement of the useful ion yield for lipids was observed and clear enhancement of image contrast for lipids in brain tissue was demonstrated. The study shows that the layer thickness has a great influence on the emission of secondary ions, and therefore, its precise control is important for optimal yield enhancement.