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

Abstract

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 18 O-cholesterol and 15 N-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.

Toward Understanding the Subcellular Distributions of Cholesterol and Sphingolipids Using High-Resolution NanoSIMS Imaging

“…In a subsequent publication, we modified the depth correction approach so that it captures the changes in cell morphology induced by lateral variations in the rate of sample erosion during NanoSIMS depth profiling. 3 This new approach, which we call the differential sputter rate (DSR) strategy, first uses the secondary electron intensities to independently construct a separate topography matrix for each secondary electron image in the depth profile. Construction of each of these topography matrices is analogous to that used in the CSR morphology reconstruction strategy, but with modifications that improved accuracy and expedited matrix construction.…”

“…L.KraftM. L. Brunet, M. A. Gorman, B. L. Kraft, M. L. 2022161622116233 First strategy for constructing visually accurate 3D SIMS depth profiling images of intracellular component distributions that does not require complementary measurements or other data that would prevent its application to existing SIMS depth profiling data sets .…”

“…Copyright 2016 American Vacuum Society. Reprinted with permission from ref . Copyright 2022 American Chemical Society.…”

“…(See text for details.) Reprinted with permission from ref . Copyright 2022 American Chemical Society.…”

Next Generation of Mass Spectrometry Imaging: from Micrometer to Subcellular Resolution

“…Thus, the DESI-MSI method has mainly been made available for tissue imaging. Nano secondary ion mass spectroscopy (Nano-SIMS) can obtain the best spatial resolution down to 50 nm among all the MSI methods. − Nevertheless, the utilization of a high-energy ion beam desorption/ionization source (∼keV MeV) will induce serious ion fragmentation, restricting its employment only for the analysis of elements. This problem is mitigated by time-of-flight secondary ion mass spectroscopy (ToF-SIMS) technology, but the spatial resolution is also sacrificed, making its optimal value about 1 μm. − Based on the laser beam desorption/ionization source, different MSI methodologies displayed multiscale spatial resolution.…”