Paper AS-TuM9
ToF-SIMS Dual Beam Depth Profiling and Imaging of Human HeLa Cells

Tuesday, October 21, 2008, 10:40 am, Room 207

ToF-SIMS dual beam depth profiling is commonly used in the semiconductor industry to characterize ultra thin inorganic layers with high lateral and depth resolutions (i.e., 300 and 1 nm). This technique, which combines a low fluence ion beam for analysis and a high fluence ion beam for sputtering, is now available to study organic and biological materials using polyatomic primary ions (e.g. Bi₃⁺ and C₆₀⁺). However, the more complex and fragile biological materials require special sampling handling to get relevant ToF-SIMS 3D images under UHV conditions. Also, the large ToF-SIMS 3D image data sets present challenges for analysis and interpretation. In this study, human HeLa cells were seeded onto PET substrates, then ToF-SIMS dual beam depth profiles and images of the cells were obtained using 25 keV Bi₃⁺ (analysis) and 20 keV C₆₀⁺ (sputtering) primary ions. Different Bi/ C₆₀ ion fluences and sample preparation methods (e.g., chemical fixation in 4% PBS paraformaldehyde, vitrification in trehalose, snap-freezing, cryomicrotome sectioning, etc.) were compared and the results were interpreted using principal component analysis. As an example, our results show that for the HeLa cells fixed in 4% PBS paraformaldehyde on PET, 1x10¹⁴ C₆₀⁺ ions per cm² are necessary to remove the surface contamination layer so the cell structure could be imaged. After this contamination removal, the high resolution secondary ion images obtained using Bi₃⁺ primary ions show that morphology of cells is preserved and that intracellular structures can be distinguished and chemically mapped. Our results also show that the interface between the cytoplasm and the PET substrate is reached after 2x10¹⁴ C₆₀⁺ ions per cm² while 4x10¹⁴ C₆₀⁺ ions per cm² are necessary to reach the interface between the nucleus and the PET. This result indicates that the cell nucleus is significantly thicker than the cytoplasm. Finally, our results show that the intensity of characteristic cell peaks (i.e., the phosphocholine head group from the cell membrane at m/z=184) decrease strongly with the increasing Bi₃⁺ fluence, indicating that the analysis beam fluence must be carefully controlled to avoid significant chemical damage during ToF-SIMS dual beam depth profiling.