Felix Kollmer

Analysis of Lung Surfactant Model Systems with Time-of-Flight Secondary Ion Mass Spectrometry

An often-used model lung surfactant containing dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and the surfactant protein C (SP-C) was analyzed as Langmuir-Blodgett film by spatially resolved time-of-flight secondary ion mass spectrometry (TOF-SIMS) to directly visualize the formation and composition of domains. Binary lipid and lipid/SP-C systems were probed for comparison. TOF-SIMS spectra revealed positive secondary ions (SI) characteristic for DPPC and SP-C, but not for DPPG. SI mapping results in images with domain structures in DPPC/DPPG and DPPG/SP-C, but not in DPPC/SP-C films. We are able to distinguish between the fluid and condensed areas probably due to a matrix effect. These findings correspond with other imaging techniques, fluorescence light microscopy (FLM), scanning force microscopy (SFM), and silver decoration. The ternary mixture DPPC/DPPG/SP-C transferred from the collapse region exhibited SP-C-rich domains surrounding pure lipid areas. The results obtained are in full accordance with our earlier SFM picture of layered protrusions that serve as a compressed reservoir for surfactant material during expansion. Our study demonstrates once more that SP-C plays a unique role in the respiration process.

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.

Spatial-Resolution Limits in Mass Spectrometry Imaging of Supported Lipid Bilayers and Individual Lipid Vesicles

The capabilities of time-of-flight secondary ion mass spectrometry (TOF-SIMS) with regards to limits in lateral resolution for biological samples are examined using supported lipid bilayers and individual lipid vesicles, both being among the most commonly used cell membrane mimics. Using supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers confined to a SiO(2) substrate by a chemically modified gold surface, the edge of the lipid bilayer was analyzed by imaging TOF-SIMS to assess the lateral resolution. The results using 80 keV Bi(3)(2+) primary ions show that, under optimized conditions, mass spectrometry imaging of specific unlabeled lipid fragments is possible with sub-100 nm lateral resolution. Comparison of the secondary ion yields for the phosphocholine ion (m/z 184) from a POPC bilayer using C(60)(+) or Bi(3)(+) primary ions showed similar results, indicating an advantage of Bi(3)(+) primary ions for high-resolution imaging of lipid membranes, due to their better demonstrated focusing capability. Moreover, using 300 nm vesicles of different lipid composition, the capability to detect and chemically identify individual submicrometer lipid vesicles at separations down to approximately 1 microm is demonstrated.

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.

In-Situ TOF-SIMS and SFM Measurements Providing True 3D Chemical Characterization of Inorganic and Organic Nanostructures

Information on the chemical composition, physical properties and the three dimensional structure of materials and devices at the nanometer scale is of major importance in nanoscience and nanotechnology. Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) is known to be an extremely sensitive surface imaging technique which provides elemental as well as comprehensive molecular information on all types of solid surfaces. Depth profiling of multilayers with high depth resolution as well as threedimensional analysis is performed in the so-called dual beam mode. In this mode the pulsed analysis beam is combined with a low energy sputter ion beam for the removal of material providing chemical 3D information of the sample. However, the topography of the initial sample surface as well as the subsequent evolution of the topography due to different erosion rates of the compounds cannot be identified by the technique and lead to distortions of the detected depth distribution. In order to get a true image of the 3D volume, the time scale of the TOF-SIMS 3D depth profiles needs to be converted into a depth scale. Scanning Force Microscopy (SFM) provides the required complementary information on the surface topography with a resolution on the nanometer level. Beyond that SFM can provide valuable information about the physical properties of the sample if the cantilever is operated in the different dynamic operation modes.

TOF-SIMS Analysis with High Lateral and High Mass Resolution in Parallel

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a very sensitive surface analytical technique. It provides detailed elemental and molecular information about surfaces, thin layers, interfaces, and full three-dimensional analysis of the sample. A general strength of the applied time-offlight mass analyzer is the very high transmission that is due to the fact that the entire mass range is analyzed in parallel. Any selection of peaks prior to the analysis is not required.

SFM Assisted In-Situ by ToF-SIMS: Accessing Chemical Information in True Three Dimensions

Scanning Force Microscopy (SFM) is a well-established tool for surface analysis in research and industry around the world nowadays. Originally developed to image atoms, the range of applications has since been widely deployed to make it not only an imaging technique but also a sensor of local forces of the surface and a manipulator of atoms and molecules. Very sharp, functionalized tips mounted on micro-levers constitute extremely sensitive sensors to the local force field emanating from the surface and thus make the SFM a general tool for the analysis of physical properties at the nanoscale.