We report a means by which atomic and molecular secondary ions, including cholesterol and fatty acids, can be sputtered through single-layer graphene to enable secondary ion mass spectrometry (SIMS) imaging of untreated wet cell membranes in solution at subcellular spatial resolution. We can observe the intrinsic molecular distribution of lipids, such as cholesterol, phosphoethanolamine and various fatty acids, in untreated wet cell membranes without any labeling. We show that graphene-covered cells prepared on a wet substrate with a cell culture medium reservoir are alive and that their cellular membranes do not disintegrate during SIMS imaging in an ultra-high-vacuum environment. Ab initio molecular dynamics calculations and ion dose-dependence studies suggest that sputtering through single-layer graphene occurs through a transient hole generated in the graphene layer. Cholesterol imaging shows that methyl-β-cyclodextrin preferentially extracts cholesterol molecules from the cholesterol-enriched regions in cell membranes.
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The data that support the findings of this study are available in figshare at https://doi.org/10.6084/m9.figshare.13356740. We provide the raw data for Fig. 2 calculated using VASP and imaged with VESTA, and the ToF-SIMS data for Figs. 1, 3 and 4 in the ITA format for spectra and images. ToF-SIMS data can be opened and analyzed using ION-TOF SurfaceLab software. Source data are provided with this paper.
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This work was supported by the DGIST R&D Program (18-BD-06) and the Basic Research Program (2016R1A2B4009037) of the Ministry of Science and ICT, Korea, and the National Research Foundation of Korea (NRF-2017R1A4A1015534 and 2018R1A2A3075499). Computing time was supported by the KISTI Grand Challenge Program (KSC-2017-C3-0065).
The authors declare no competing interests.
Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended Data Fig. 1 Fabrication of a cell culture wet substrate.
a, Low-stress silicon nitride (Si3N4) of 100 nm thickness was deposited on both sides of a silicon wafer. b, The Si3N4 thin film on the front side was patterned into 2 × 2 or 3 × 3 array of 40 × 40 microholes with each 1 μm hole spaced at 3–4 μm. c, The Si3N4 thin film on the back side was formed into a large window of 2.5 × 2.5 mm2 in the center. d, Photoresist (PR) was stripped off from both sides. e, The back side of the silicon wafer was etched to expose the microhole array in the center of the Si3N4 membrane. f, Collagen film was coated on the front side of the wafer. g, Following the injection of a drop of cell culture medium into the hollow cavity, an HDPE film was placed on the drop. h, The cell culture medium substrate was sealed by applying UV-curing optical adhesive between the back side of the wafer and a glass substrate. i, Cells were cultured on the collagen film over the microhole array followed by a graphene capping onto the cells. j, k, The front (j) and back (k) sides of the patterned substrate with a microhole array Si3N4 membrane. l, Collagen film coated on the top of the substrate. m–o, a microhole array Si3N4 membrane. To increase the success rate of obtaining SIMS images without microcracks in graphene, we fabricated four sets of 3 × 3 array of 40 × 40 μm2 microholes on a cell culture substrate (m).
Extended Data Fig. 2 ToF-SIMS images of graphene-covered untreated wet A549 cells.
a–j, ToF-SIMS positive and negative ion images of a cholesterol fragment at C7H11+, m/z 95.07 (a), myristic acid at C14H27O2−, m/z 227.22 (b), palmitoleic acid at C16H29O2−, m/z 253.23 (c), linoleic acid at C18H31O2−, m/z 279.25 (d), phosphocholine fragment at C5H13NPO3+, m/z 166.05 (e), phosphocholine at C5H15NPO4+, m/z 184.04 (f), H3O+ at m/z 19.02 (g), OH− at m/z 17.00 (h), Na+ at m/z 22.99 (i), and K+ at m/z 38.96 (j). Additional secondary ion images for Fig. 1 are provided here for extra information. Scale bar, 100 μm (a–j). Results presented are obtained from a single experiment.
Extended Data Fig. 3 Cell viability assay.
a, Merged image with fluorescent and bright-field optical images of graphene-covered A549 cells on a wet substrate that were killed in 70% methanol for 30 min shows that EthD-1 dye molecules can migrate from a culture media reservoir to the dead cells in the vicinity of the microholes. b–e, Fluorescence images of the graphene-covered cells on a wet substrate from 5–70 min, respectively, after graphene capping without exposure to vacuum. No cell death signal from the cells in the vicinity of the microholes was observed. f–j, Fluorescence images of the graphene-covered cells on a wet substrate from 40–70 min, respectively, after graphene capping with exposure to the vacuum of 1 x 10−5 mbar for 5 min. The merged image in Fig. f with fluorescent and bright-field optical images obtained 40 min after the graphene capping shows the location of a microhole array indicated by the colored dash-lines in g–j and Fig. 1i,j. No cell death signal from the cells in the vicinity of the microholes was observed, suggesting that the cellular membrane does not disintegrate for over 60 min after the graphene capping with the exposure to the vacuum. Green and magenta here indicate live and dead cells before graphene capping, respectively. Scale bar, 50 μm (a–j). Results presented are obtained from a single experiment.
Extended Data Fig. 4 Conventional ToF-SIMS imaging of fixed and dried A549 cells.
a–b, Optical images and ToF-SIMS images of (left to right) phosphocholine, cholesterol, palmitic acid, Na+ and H3O+ for formalin-fixed (a) and osmium tetroxide-fixed A549 cells (b). The detailed distributions of lipids are not visible in the ToF-SIMS images of fixed and dried cells in contrast to Fig. 1, which suggests a significant distortion of the observed lipids images from the intrinsic distribution due to the fixation and drying. The H3O+ intensity from the osmium tetroxide-fixed cells (b) is lower than the background. Scale bar, 100 μm (a–b). Results presented are obtained from a single experiment.
Extended Data Fig. 5 Primary ion dose dependence of Si+ secondary ion intensity of a cover glass with graphene-covered fixed A549 cells.
a, ToF-SIMS Si+ ion images for a cover glass with graphene-covered fixed A549 cells analyzed with four different ion doses: (left to right) 1.3 × 1013, 1.3 × 1014, 1.3 × 1015, and 1.3 × 1016 ions/cm2. b, HIM images of the bombarded regions of the samples corresponding to ToF-SIMS images in a show no visible large-scale defects in graphene due to ion beam bombardment of at least up to 1.3 × 1014 ions/cm2; however, the ion doses more than 1.3 × 1015 ions/cm2 etch the graphene away. c–d, ToF-SIMS Si+ image counts depending on the ion doses of up to 1.3 × 1015 (c) and 1.3 × 1016 ions/cm2 (d) reveal no ion dose dependence of secondary ion intensity within the ion dose of 1014 ions/cm2, thus indicating that the sputtering through single-layer graphene is not due to the defects generated by the ion beam bombardment. The SIMS images were taken for the ion dose much less than 1014 ions/cm2. The large-scale defects in the HIM images in b are due to the cracks in the graphene layer on the cells. Wet cells under graphene with cracks were generally not used for SIMS imaging. Scale bar, 5 μm (a–b). Results presented are obtained from a single experiment.
Extended Data Fig. 6 HIM and Raman analysis of the bombarded region of graphene-covered fixed HDF cells.
a–c, HIM images of the bombarded region of the sample. The bombarded region appears to be a dark square indicated by white-dotted lines in ToF-SIMS analysis (a). The appearance of the dark spots for each pixel suggests the decrease of secondary electron yields in the HIM imaging due to an increase of the work function with the graphene oxide formation (b). Magnified HIM image of one bombarded dark spot shows no noticeable damage in graphene at the 1.93 × 1014/cm2 ion dose (c). d, ToF-SIMS total negative ion image of the sample corresponding to the HIM image in a. e, Raman analysis of the ion-bombarded region of graphene clearly shows the formation of graphene oxide in contrast to the non-bombarded region of pristine graphene. The graphene oxide may be formed by the ion-beam-induced reaction between graphene and water molecules beneath the graphene. Results presented are obtained from a single experiment.
Extended Data Fig. 7 Cholesterol images with high intensity.
a, d, ToF-SIMS positive ion images of cholesterol at C27H45+, m/z 369.25 over areas of 500 x 500 μm2 (a) and 150 × 150 μm2 (d). b–c, e–f, Cholesterol images with pixels above a certain threshold (10 for b, c and 4 for e, f) per pixel in the images of a and d. Gradation was removed for clarity in c and f. The heterogeneous images in c and f are similar to the image reported by nano-SIMS21 using cholesterol antibodies that work for a high cholesterol concentration, which indicates that the cholesterol images in a and d show the intrinsic whole cholesterol distribution. Scale bar, 100 μm (a–c) and 30 μm (d–f). Results presented are obtained from a single experiment.
Cell viability study using optical imaging. The cell shape and the intracellular structure in the vicinity of microholes 15 min after graphene capping without exposure to vacuum did not show any drastic change; however, internal vesicle motions slowed by 10 min. A wet substrate was made of a silicon dioxide (SiO2) membrane with a thickness of ~100 nm and an area of ~2 × 2 mm2 to obtain clear optical images. However, this configuration caused a fracture of the large, thin membrane in the wet substrate due to a large strain in the vacuum.
Source Data Fig. 1
Source Data for Fig. 4e,f
Source Data Extended Data Fig. 1
Source Data for Extended Data Fig. 5c,d
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Lim, H., Lee, S.Y., Park, Y. et al. Mass spectrometry imaging of untreated wet cell membranes in solution using single-layer graphene. Nat Methods 18, 316–320 (2021). https://doi.org/10.1038/s41592-020-01055-6
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