M/Z: 585.3149

The Bathymodiolus puteoserpentis specimen used for high resolution AP-MALDI-MSI was collected during the RV Meteor M126 cruise in 2016 at the Logatchev hydrothermal vent field on the Mid-Atlantic Ridge. The specimen was retrieved with the MARUM-Quest remotely operated vehicle (ROV) at the Irina II vent site at 3038 m depth, 14°45’11.01”N and 44°58’43.98”W, and placed in an insulated container to prevent temperature changes during recovery. Gills were dissected from the mussel as soon as brought on board after ROV retrieval, submerged in precooled 2% w/v carboxymethyl cellulose gel (CMC, Mw ~ 700,000, Sigma-Aldrich Chemie GmbH) and snap-frozen in liquid N2. Samples were stored at -80 °C until use.
The CMC-embedded gills were cross-sectioned at 10 µm thickness with a cryostat (Leica CM3050 S, Leica Biosystems Nussloch GmbH) at a chamber temperature of -35 °C and object holder at -22 °C. Individual sections were thaw-mounted onto coated Polysine slides (Thermo Scientific) and subsequently frozen in the cryostat chamber. Slides with tissue sections were stored in slide containers with silica granules, to prevent air moisture condensation on the tissue upon removal from the freezer. Before AP-MALDI matrix application, the sample was warmed to room temperature under a dry atmosphere in a sealed slide container (LockMailer microscope slide jar, Sigma-Aldrich, Steinheim, Germany), filled with silica granules (Carl Roth GmbH) to avoid condensation on the cold glass slide. The sample glass slide was marked with white paint around the tissue for orientation during image acquisition as previously described[1]. Additionally, optical images of the tissue section were acquired with a digital microscope (VHX-5000 Series, Keyence, Neu-Isenburg, Germany) prior to matrix application. To apply the matrix, we used an ultrafine pneumatic sprayer system with N2 gas (SMALDIPrep, TransMIT GmbH, Giessen, Germany)[2], to deliver 100 μl of a 30 mg/ml solution of 2,5-dihydroxybenzoic acid (DHB; 98% 574 purity, Sigma-Aldrich, Steinheim, Germany) dissolved in acetone/water (1:1 v/v) containing 0.1% trifluoroacetic acid (TFA). To locate the field of view and facilitate laser focusing, a red marker was applied adjacent to the matrix-covered tissue section. Ref: [1] Kaltenpoth M, Strupat K, Svatoš A Linking metabolite production to taxonomic identity in environmental samples by (MA)LDI-FISH. ISME J. 2016 Feb;10(2):527-31. doi: 10.1038/ismej.2015.122. PMID:26172211 [2] Kompauer M, Heiles S, Spengler B. Atmospheric pressure MALDI mass spectrometry imaging of tissues and cells at 1.4-μm lateral resolution. Nat Methods. 2017 Jan;14(1):90-96. doi: 10.1038/nmeth.4071. PMID:27842060
High-resolution AP-MALDI-MSI measurements were carried out at an experimental ion source setup [1][2], coupled to a Fourier transform orbital trapping mass spectrometer (Q Exactive HF, Thermo Fisher Scientific GmbH, Bremen, Germany). The sample was rastered with 233 x 233 laser spots with a step size of 3 µm without oversampling, resulting in an imaged area of 699 x 699 µm. AP-MALDI-MSI measurements were performed in positive mode for a mass detection range of 400–1200 Da and a mass resolving power of 240,000 (at 200 m/z). After AP-MALDI-MSI, the measured sample surface was recorded using a stereomicroscope (SMZ25, Nikon, Düssedorf, Germany). Ref: [1] Kompauer M, Heiles S, Spengler B. Atmospheric pressure MALDI mass spectrometry imaging of tissues and cells at 1.4-μm lateral resolution. Nat Methods. 2017 Jan;14(1):90-96. doi: 10.1038/nmeth.4071. PMID:27842060 [2] Kompauer M, Heiles S, Spengler B. Autofocusing MALDI mass spectrometry imaging of tissue sections and 3D chemical topography of nonflat surfaces. Nat Methods. 2017 Dec;14(12):1156-1158. doi:10.1038/nmeth.4433. PMID:28945703

1 male adult wild-type rat was obtained from Inserm U1085 - Irset Research Institute (University of Rennes1, France). Animals were age 60 days and were reared under ad-lib conditions. Care and handling of all animals complied with EU directive 2010/63/EU on the protection of animals used for scientific purposes. The whole epididymis was excised from each animal immediately post-mortem, loosely wrapped rapidly in an aluminum foil and a 2.5% (w/v) carboxymethylcellulose (CMC) solution was poured to embed the epididymis to preserve their morphology. To remove air bubbles, the filled aluminum molds was gently freezed by depositing it on isopentane or dry ice, then on the nitrogen vapors and finally by progressively dipping the CMC/sample coated with aluminum foil into liquid nitrogen (or only flush with liquid nitrogen). Frozen tissues were stored at -80 °C until use to avoid degradation.

Fig. 2 MALDI-MSI data from the same mouse lung tissue analyzed in Fig. 1. A: Optical image of the post-MSI, H&E-stained tissue section. B–D, F–G: Ion images of (B) m/z 796.6855 ([U13C-DPPC+Na]+), (C) m/z 756.5514 ([PC32:0+Na]+), (D) m/z 765.6079 ([D9-PC32:0+Na]+), (F) m/z 754.5359 ([PC32:1+Na]+), and (G) m/z 763.5923 ([D9-PC32:1+Na]+). E, H: Ratio images of (E) [D9-PC32:0+Na]+:[PC32:0+Na]+ and (H) [D9-PC32:1+Na]+:[PC32:1+Na]+. Part-per-million (ppm) mass errors are indicated in parentheses. All images were visualized using total-ion-current normalization and using hotspot removal (high quantile = 99%). DPPC = PC16:0/16:0. U13C-DPPC, universally 13C-labeled dipalmitoyl PC; PC, phosphatidylcholine; MSI, mass spectrometry imaging; H&E, hematoxylin and eosin. Fig 1-3, Fig S1-S3, S5

Supplementary Figure S8. MALDI-MSI data of mouse lung tissue administered with D9-choline and U 13C-DPPC–containing Poractant alfa surfactant (labels administered 18 h prior to sacrifice). Ion images of (a) m/z 796.6856 ([U13C-DPPC+Na]+), (b) m/z 756.5154 [PC32:0+Na]+ and (c) m/z 765.6079 ([D9-PC32:0+Na]+). (d) Overlay image of [U13C-DPPC+Na]+ (red) and [D9-PC32:0+Na]+ (green). Parts per million (ppm) mass errors are indicated in parentheses. All images were visualised using totalion-current normalisation and using hotspot removal (high quantile = 99%). DPPC = PC16:0/16:0.

Supplementary Figure S6. Ion distribution images for (a) [PC36:4+Na]+ (m/z 804.5514) and (b) [PC38:6+Na]+ (m/z 828.5515) obtained from mouse lung tissue collected 6 h after administration of D9- choline and U13C-DPPC–containing CHF5633. Parts-per-million (ppm) mass errors are indicated in parentheses. (c) Magnification of the boxed region in (a) with selected bronchiolar regions outlined in white boxes. (d) The corresponding H&E-stained tissue section with the same selected bronchiolar regions outlined in black boxes. These data demonstrate the co-localisation of the polyunsaturated lipids PC36:4 and PC38:6 with the bronchiolar regions of the lung. All MSI images were visualised using total ion current normalisation and hotspot removal (high quantile = 99%).

single cell layer class_4 is the gill structure cells, metabolite ion 534.2956 is the top representive ion of this type of cell