Depth Correction of 3D NanoSIMS Images Shows Intracellular Lipid and Cholesterol Distributions while Capturing the Effects of Differential Sputter Rate

Knowledge of the distributions of drugs, metabolites, and drug carriers within cells is a prerequisite for the development of effective disease treatments. Intracellular component distribution may be imaged with high sensitivity and spatial resolution by using a NanoSIMS in the depth profiling mode. Depth correction strategies that capture the effects of differential sputtering without requiring additional measurements could enable producing accurate 3D NanoSIMS depth profiling images of intracellular component distributions. Here we describe an approach for depth correcting 3D NanoSIMS depth profiling images of cells that accounts for differential sputter rates. Our approach uses the secondary ion and secondary electron depth profiling images to reconstruct the cell's morphology at every raster plane. These cell morphology reconstructions are used to adjust the z-positions and heights of the voxels in the component-specific 3D NanoSIMS images. We validated this strategy using AFM topography data and reconstructions created from depth profiling images acquired with focused ion beam-secondary electron microscopy. Good agreement was found for the shapes and relative heights of the reconstructed morphologies. Application of this depth correction strategy to 3D NanoSIMS depth profiling images of a metabolically labeled cell better resolved the transport vesicles, organelles, and organellar membranes containing 18O-cholesterol and 15N-sphingolipids. Accurate 3D NanoSIMS images of intracellular component distributions may now be produced without requiring correlated analyses with separate instruments or the assumption of a constant sputter rate. This will allow visualization of the subcellular distributions of lipids, metabolites, drugs, and nanoparticles in 3D, information pivotal to understanding and treating disease.