Paper BI2+AS-TuA12
Molecular Depth Profiling and 3D Imaging of Biological Samples by ToF-SIMS: From Model Amino Acid Films to Real Biological Cells

Tuesday, October 19, 2010, 5:40 pm, Room Taos A

Time-of-Flight Secondary Ion Mass Spectrometry is now routinely used to obtain molecular information about the surface of complex biological samples and biomaterials. The recent advent of cluster ion beams such as Bi₃⁺ and C₆₀⁺ provides enhanced sensitivity for high mass fragments, which in turn allows 2D imaging of specific biomolecules with improved sensitivity and sub-micrometer lateral resolution. Molecular depth profiling of biological samples with low chemical damage and high depth resolution (i.e., < 10 nm) has also been shown to be possible by etching with cluster projectiles such as C₆₀ and with low energy cesium ions. Since ToF-SIMS also allows the detection of all elements without the need of specific markers, the technique shows great potential for molecular 3D imaging of single cells and could become an inevitable complementary tool to MALDI and fluorescence microscopy for biomedical research in the near future.

However, only few examples of full 3D images of biological cells have been reported in the literature at this time. This limitation probably comes from the facts that preparing the cells for chemical analysis under UHV environment is tedious, and that our understanding of energetic primary ions/biological matter interactions is limited. Fundamental studies of these aspects are indeed difficult during 3D imaging because the cells are complex, heterogeneous, non-flat samples with relatively unknown molecular composition.

In this work, fundamental biological matter/projectile interactions were studied by depth profiling model amino acid films under different ToF-SIMS conditions. The influence of the experimental parameters on the quality of the depth profiles was investigated by measuring the sputter rates, the depth resolutions and the intensities of the molecular ion signals with respect to the chemistry of the target (e.g., arginine vs phenylalanine), the nature of the primary ion species (e.g., C₆₀⁺ vs Cs⁺) and the bombardment conditions (e.g., fluence and energy). Then the complexity of the model samples was increased by mixing several amino acids and by creating multilayer films. This approach was another step toward the analysis of real biological samples. Again, ToF-SIMS fundamentals were investigated by measuring, e.g., the interface widths between the different organic layers and the signal decay due to the increasing etching fluence. Finally, optimal 3D images of single HeLa cells were acquired and were discussed based on the data obtained for the model amino acid films. The effect of the sample preparation on the quality of the images was also investigated.