Lipid metabolism and intracellular transport regulate the relative abundances of lipids and cholesterol within organelles. Disturbances in lipid metabolism and transport may produce abnormal lipid distributions between organelles within a cell, and they are hallmarks of some diseases. Three-dimensional (3D) images that show the distributions of rare isotope-labeled cholesterol and lipid species of interest may be generated by imaging the cell with a Cameca NanoSIMS instrument operated in a depth profiling mode and rendering the resulting stack of individual depth profiling images. However, identification of the organelles that contain the components of interest can be challenging because the resulting 3D NanoSIMS depth profiling images are distorted in the z-direction. To address this issue, we have developed a strategy that corrects this image distortion in the z-direction without requiring the acquisition of topography data or collection of substrate-specific secondary ions. Instead, the secondary electron images that are acquired during NanoSIMS depth profiling are used to construct a model of the cell’s morphology. This morphology reconstruction is then used to shift the voxels within the 3D NanoSIMS depth profiling images to the correct position. Application of this approach to depth correct 3D NanoSIMS depth profiling images of a metabolically labeled mammalian cell yielded more accurate representations of the cholesterol and sphingolipid distributions in the cell. Depth correction improved the clarity of the component-specific 3D images, allowing transport vesicles and organellar membranes containing rare isotope labeled cholesterol and sphingolipids to be more clearly visualized.

Construction of accurate 3D images of intracellular cholesterol and sphingolipid distributions enabled by depth correction of NanoSIMS depth profiling images

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