Observation of endoplasmic reticulum tubules via TOF-SIMS tandem mass spectrometry imaging of transfected cells

Abstract

Advances in three-dimensional secondary ion mass spectrometry (SIMS) imaging have enabled visualizing the subcellular distributions of various lipid species within individual cells. However, the difficulty of locating organelles using SIMS limits efforts to study their lipid compositions. Here, the authors have assessed whether endoplasmic reticulum (ER)-Tracker Blue White DPX, which is a commercially available stain for visualizing the endoplasmic reticulum using fluorescence microscopy, produces distinctive ions that can be used to locate the endoplasmic reticulum using SIMS. Time-of-flight-SIMS tandem mass spectrometry (MS) imaging was used to identify positively and negatively charged ions produced by the ER-Tracker stain. Then, these ions were used to localize the stain and thus the endoplasmic reticulum, within individual human embryonic kidney cells that contained higher numbers of endoplasmic reticulum-plasma membrane junctions on their surfaces. By performing MS imaging of selected ions in parallel with the precursor ion (MS) imaging, the authors detected a chemical interference native to the cell at the same nominal mass as the pentafluorophenyl fragment from the ER-Tracker stain. Nonetheless, the fluorine secondary ions produced by the ER-Tracker stain provided a distinctive signal that enabled locating the endoplasmic reticulum using SIMS. This simple strategy for visualizing the endoplasmic reticulum in individual cells using SIMS could be combined with existing SIMS methodologies for imaging intracellular lipid distribution and to study the lipid composition within the endoplasmic reticulum.

Identification and High-Resolution Imaging of α-Tocopherol from Human Cells to Whole Animals by TOF-SIMS Tandem Mass Spectrometry

“…Importantly, the demonstrated capability for TOF-SIMS tandem MS imaging can be applied to assess the localization of a vast range of membrane-associated, as well as intracellular molecules and lipids [ 33 ], providing a means to confidently establish molecular identification and to visualize those same molecules which was not previously possible, also improving spatial resolution over previously available techniques. Specifically, mapping the spatial distribution of a molecule like α-tocopherol in 2- and 3-dimensions as in this study, using both thin tissue sections and cultured cells, demonstrates a unique and valuable tool to investigate the role of oxidative stress in disease states.…”

Demystifying and Unravelling the Factual Molecular Structure of the Biopolymer Sporopollenin

“…A detailed description of this TOF-TOF instrument has been reported previously, including the attained spatial resolution, monoisotopic precursor selection, and kilo electron volt collision-induced dissociation (keV-CID) of the selected precursor ions to generate the tandem MS product ion spectra . [52][53][54] The elements and qualities of the tandem MS spectra for composition analysis and structure elucidation have been further elaborated in other reports . [55,56] In the present study, all the spectra and images were recorded with electrodynamically bunched pulses of a 30 keV Bi3+ primary ion beam of which the DC current was measured to be ≈ 9 nA.…”

Imaging the Endoplasmic Reticulum within Individual Mammalian Cells with Secondary Ion Mass Spectrometry

“…Time-of-flight-SIMS (TOF-SIMS) tandem MS imaging confirmed the ER-Tracker stain produced fluoride and other negatively charged ions, including a pentafluorophenyl anion [C6F5 -], that were characteristic of the stain (Figure 1) [5]. TOF-SIMS imaging of the fluoride ions permitted locating the ER-tracker stain, and thus, the endoplasmic reticulum tubules within the cell (Figure 2) [5]. We expect that combining this stain can be combined with strategies for detecting lipid species of interest would enable probing the lipid composition within the endoplasmic reticulum.…”

“…To minimize the amount of sputtering that would be required to expose the endoplasmic reticulum within the cell to the analysis beam, we used a cell line in which the endoplasmic reticulum formed a relatively high number of tubular junctions with the plasma membrane for this study [4]. Time-of-flight-SIMS (TOF-SIMS) tandem MS imaging confirmed the ER-Tracker stain produced fluoride and other negatively charged ions, including a pentafluorophenyl anion [C6F5 -], that were characteristic of the stain (Figure 1) [5]. TOF-SIMS imaging of the fluoride ions permitted locating the ER-tracker stain, and thus, the endoplasmic reticulum tubules within the cell (Figure 2) [5].…”