RE-CONSTRUCTION OF A 3D TOF-SIMS IMAGE OF A SINGLE BIOLOGICAL CELL FROM 2D IMAGE STACKS USING THE Z-CORRECTION METHOD

Shin Muramoto, Michael Robinson, Dan Graham, and David G. Castner

With the advent of cluster primary ions, time-of-flight secondary ion mass spectrometry (ToF-SIMS) has become one of few techniques capable of constructing 3-dimensional chemical maps with molecular sensitivity and depth resolution approaching the nanometer level.  This attribute has attracted the analysis of increasingly complex organic systems from fields such as medicine, an example of which involves creating a 3D map of biomarkers within a single cell for disease diagnostics.  One difficulty encountered in such analysis is in the nature of the data acquisition; SIMS depth profiles are acquired layer by layer, so samples with curvature or significant topography will appear reversed in the z-direction while the orientation of the internal contents are preserved.  The end result is an image that is difficult to interpret because the internal contents are not in alignment with the outer cellular features, in addition to the outer cellular structure not resembling the original shape.

In this study, we have successfully applied the z-correction pixel shift to reconstruct a 3D image of a 3T3 fibroblast, with the internal contents in alignment with the outer cellular features.  As ToF-SIMS is a vacuum technique, the cells were first chemically fixed using a 4% paraformaldehyde solution to preserve cellular structures, then depth profiled in the SIMS dual-beam mode using Bi₃⁺ analysis and C₆₀⁺⁺ sputter beams (incident energies of 25 keV and 20 keV, respectively).  Z-correction was performed using a script written in-house, and the opacity of the outer cellular features was reduced to visualize the internal cellular components.  Although small molecules were unable to be visualized due to damage accumulation, larger components such as the nuclear membrane and nucleoli were clearly visible and localized in space consistent with confocal imaging data.  In addition, AFM images of the cell cross-sections matched perfectly to the z-corrected SIMS image which suggested two things: the concept of pixel shift can be successfully applied to reconstruct a 3-dimensional image; and the sputter rate through a biological cell is constant, overturning previous claims suggesting otherwise (the sputter rate was found to be roughly 10 nm per 1 x 10¹³ C₆₀⁺⁺ ions).  The concept of z-correction lays the groundwork necessary for developing a diagnostic tool to detect disease progression by localizing in 3-dimensions the presence of key biomarkers such as lipids, metabolites, drugs, or even nanoparticles within a cell.