Subcellular Mass Spectrometry Imaging and Absolute Quantitative Analysis across Organelles

Abstract

Mass spectrometry imaging is a field that promises to become a mainstream bioanalysis technology by allowing the combination of single-cell imaging and subcellular quantitative analysis. The frontier of single-cell imaging has advanced to the point where it is now possible to compare the chemical contents of individual organelles in terms of raw or normalized ion signal. However, to realize the full potential of this technology, it is necessary to move beyond this concept of relative quantification. Here we present a nanoSIMS imaging method that directly measures the absolute concentration of an organelle-associated, isotopically labeled, pro-drug directly from a mass spectrometry image. This is validated with a recently developed nanoelectrochemistry method for single organelles. We establish a limit of detection based on the number of isotopic labels used and the volume of the organelle of interest, also offering this calculation as a web application. This approach allows subcellular quantification of drugs and metabolites, an overarching and previously unmet goal in cell science and pharmaceutical development.

Chemical Analysis of Single Cells and Organelles

“…ACS Nano . 2020 , 14 , 4316 (ref ( 145 )). Copyright 2020 American Chemical Society, and further permissions related to the material excerpted should be directed to the ACS.…”

“…In a recent paper, Thomen et al presented a novel approach to directly measure the absolute concentration of an isotopically labeled drug directly from a NanoSIMS image. 145 In addition, the optimization of NanoSIMS parameters, such as the secondary ion emission steady state ( Figure 8 B), was discussed. This study was also thoroughly explored in a perspective by Kraft and co-workers.…”

Metabolic Analysis at the Nanoscale with Multi‐Isotope Imaging Mass Spectrometry (MIMS)

“…In a very general sense, imaging such tracers with MIMS is analogous to metabolic imaging by positron emission tomography, but at the nanoscale rather than meso-scale. MIMS has been used in this way to assess protein turnover in single organelles, track cellular division in vivo, visualize sphingolipid rafts on the plasma membrane, and measure dopamine incorporation into dense-core vesicles, among other applications (Frisz et al, 2013;Lovrić et al, 2017;Narendra et al, 2020;Senyo et al, 2013;Steinhauser et al, 2012;Thomen et al, 2020). Additionally, metabolic labeling with MIMS has been applied to diverse biological systems, from microbes to humans (Lechene et al, 2006;Steinhauser et al, 2012;Guillermier, Fazeli, et al, 2017;Lechene, Luyten, McMahon, & Distel, 2007).…”

“…Consider labeling of DNA with 13 C-thymidine. 13 C has a natural abundance of ∼1.1%, and carbon has a concentration of ∼0.606 M in an embedded biological specimen (Thomen et al, 2020). Thus, to be detected, the 13 C in thymidine must exceed the variance of the background 13 C measurement.…”

“…Moreover, the dilution of signal due to the carbon content of plastic embedding medium can be viewed as an advantage for some MIMS applications. As recently shown by the Ewing laboratory, because the 13 C concentration in the embedding medium (e.g., Epon 812) is a known quantity, it can be used as an internal standard for the absolute quantification of a 13 C-containing metabolite embedded within it (Thomen et al, 2020). Thus, despite a lower SNR, 13 C may represent the best stable isotope for some applications.…”