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Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of 2017

Supporting Information

Water-soluble Au NCs for Multiplexed Imaging Jinan Li,ac Jing Liu,ad Zheyi Liu,a, Yuan Tan,bc Xiaoyan Liu,b Fangjun Wang*a aCAS Key Laboratory of Separation Sciences for , National Chromatographic R&A Center, Dalian Institute of , Chinese Academy of Sciences (CAS), Dalian 116023, China bState Key Laboratory of , iChEM, Gold Catalysis Research Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China cUniversity of Chinese Academy of Science, Beijing 100049, China dCollege of Pharmacy, Dalian Medical University, Dalian 116044, China. *E-mail: [email protected], Tel: +86-411-82464150, Fax: +86-411-84379620 Contents Page Materials and reagents S-1 Synthesis of Au NCs S-1 Experiment Synthesis of Au NPs S-1 details Materials Characterization S-1 Sample Preparation for MALDI-MS S-2 Mass spectrometry imaging. S-2 Fig. S1 (A) Illustration of the synthesis of homogeneous Au NCs; (B) Typical TEM image of Au NCs (Left) and Au NPs (Right) with scale bar of 20 -1 nm; (C) TGA results of Au NCs under N2 atmosphere with increasing temperature at 10 °C min ; (D) UV−vis absorption spectrum for as-prepared S-4 Au NCs and Au NPs. Fig. S2 Optical images of DHB crystal (A), DHB-analyte cocrystal (B), Au NCs (C), Au NCs-analyte cocrystal (D), Au NPs (E), and Au NPs-analyte S-5 cocrystal (F) on the MALDI steel plate. Fig. S3 The MS spectra of with the different concentration of Au NCs as matrix. S-6 Fig. S4 Mass spectra of carbamazepine (A, m/z = 237.9 [M+H]+, 258.8 [M+Na]+, 274.8 [M+K]+ and 280.8 [M+2Na-H]+), (B, m/z = 111.9 [M+H]+, 133.9 [M+Na]+, 149.9 [M+K]+ and 155.9 [M+2Na-H]+), (C, m/z = 307.9 [M+H]+ and 329.9 [M+Na]+) and trechalose (D, m/z = S-7 364.9 [M+Na]+) with Au NCs (a), DHB (b), Au NPs (c) as matrix, or without any matrix (d) for MALDI-MS detection in the positive- reflector mode. The amount of analyte was 5 nmol. Fig. S5 Mass spectra of (A, m/z = 172.8 [M+Na]+), trechalose (B, m/z = 364.9 [M+Na]+ and 386.9 [M+2Na-H]+), (C, m/z = 364.9 Supporting [M+Na]+, 380.9 [M+K]+ and 387.0 [M+2Na-H]+ ), (D, m/z = 172.9 [M+Na]+), (E, m/z = 527.0 [M+Na]+), (F, m/z = 364.9 Figures and [M+Na]+), (G, m/z = 364.9 [M+Na]+ and 381.0 [M+K]+), glucose (H, m/z = 202.9 [M+Na]+), (I, m/z = 202.9 [M+Na]+) and 1- S-8 Tables progargyl-O-maltose (J, m/z = 402.8 [M+Na]+) with Au NCs for MALDI-MS detection in the positive-ion reflector mode. The amount of analyte was 5 nmol. Fig. S6 Mass spectra of (A, m/z = 243.9 [M+H]+,265.9 [M+Na]+ and 281.9 [M+K]+), cytosine (B, m/z = 111.0 [M+H]+,133.9 [M+Na]+, 149.9 [M+K]+ and 155.9 [M+2Na-H]+), (C, m/z = 266.9 [M+Na]+), triolein (D, m/z = 907.8 [M+Na]+), oleic acid (E, m/z = 305.4 [M+Na]+ and 327.4 [M+2Na-H]+), (F, m/z = 303.8 [M+Na]+ and 325.4 [M+2Na-H]+), linolenic acid (G, m/z = 301.4 [M+Na]+, 323.5 [M+2Na-H]+ and S-9 339.4 [M+Na+K-H]+), (H, m/z = 279.5 [M+Na]+ and 301.5 [M+2Na-H]+) and arachidonic acid (I, m/z = 327.3 [M+Na]+, 349.6 [M+2Na- H]+ and 365.4 [M+Na+K-H]+) with Au NCs for MALDI-MS detection in the positive-ion reflector mode. The amount of analyte was 5 nmol. Fig. S7 Mass spectra of 3-O-tigloylswietenolide (A, m/z = 591.0 [M+Na]+), atropine (B, m/z =290.0 [M+H]+, 311.9 [M+Na]+ and 327.9 [M+K]+), azedarachol (C, m/z = 443.0 [M+Na]+), gastrodin (D, m/z = 309.0 [M+Na]+, 325.0 [M+K]+ and 331.0 [M+2Na-H]+) and carbamazepine (E, m/z = S-10 258.8 [M+Na]+, 274.8 [M+K]+ and 250.8 [M+2Na-H]+) with Au NCs for MALDI-MS detection in the positive-ion reflector mode. The amount of analyte was 5 nmol. Fig. S8 Mass spectra of W-G (A, m/z = 261.9 [M+H]+ and 283.9 [M+Na]+), G-W-G (B, m/z = 318.9 [M+H]+ and 341.0 [M+Na]+), W-L (C, m/z = 318.0 [M+H]+ and 340.1 [M+Na]+ ), F-A (D, m/z = 236.9 [M+H]+ and 250.9 [M+Na]+), F-A-N (E, m/z = 351.0 [M+H]+ and 373.1 [M+Na]+), F-G-G (F, m/z = 280.0 [M+H]+ and 302.0 [M+Na]+), F-E (G, m/z = 295.0 [M+H]+ and 317.0 [M+Na]+), YV (H, m/z = 281.0 [M+H]+ and 303.1 [M+Na]+) S-11 and glutathione (I, m/z = 308.0 [M+H]+ and 330.0 [M+Na]+) with Au NCs for MALDI-MS detection in the positive-ion reflector mode. The amount of analyte was 5 nmol. Fig. S9 Heatmap generated from the relative deviation of MS intensity related to the analytes commonly detected with Au NCs and DHB as matrix. S-12 Relative deviation is the difference between 1 and the ratio of MS intensity experimental value and average value Fig. S10 Mass spectra of 1-propargyl-O-maltose with Au NCs as matrix. The analyte was 5 nmol with different concentration of NaCl (i), NH HCO 4 3 S-13 (ii) and BSA (iii). The solvent is H2O, H2O and 10 mM NH4HCO3, respectively. Fig. S11 Mass spectra of F-E with Au NCs as matrix. The analyte was 5 nmol with different concentration of NaCl (i), NH HCO (ii) and 4 3 S-13 BSA (iii). The solvent is H2O, H2O and 10 mM NH4HCO3, respectively. Fig. S12 Mass spectra of cytosine with Au NCs as matrix. The analyte was 5 nmol with different concentration of NaCl (i), NH HCO (ii) and BSA 4 3 S-14 (iii). The solvent is H2O, H2O and 10 mM NH4HCO3, respectively. Fig. S13 Average MS spectra of Au NCs-assisted MSI of mouse brain slice. S-15 Fig. S14 Average MS spectra of DHB-assisted MSI of mouse brain slice. The peaks signed with × were caused by DHB. S-16 Fig. S15 MS images related to PAs, PCs and other kinds of commonly detected by both Au NCs and DHB assisted MSI. S-17 Fig. S16 MS images related to biomolecules detected only by Au NCs assisted MSI. S-18 Fig. S17 MS images related to biomolecules detected only by DHB assisted MSI. S-19 Table S1 The chemical structure, special charge of relative , detection RSD (Au NCs and DHB), and LODs (Au NCs) of analytes. S-20 Table S2 MS peak intensities and signal to noise ratios (S/N) of the seven kinds of biomolecules with Au NCs, DHB, and Au NPs as matrix, S-27 respectively. Table S3 List of biomolecules commonly detected in MSI of mouse brain by both Au NCs and DHB. The * represents that the ion was recorded with S-28 high-quality MS images (with clear lateral distribution outlines). Table S4 List of analytes detected in MSI of mouse brain by Au NCs. The * represents that the ion was recorded with high-quality MS images (with S-35 clear lateral distribution outlines). Table S5 List of analytes detected in MSI of mouse brain by DHB. The * represents that the ion was recorded with high-quality MS images (with S-39 clear lateral distribution outlines). 1. Experiment details

Materials and reagents

Cysteine (Cys), 2,5-dihyroxyl benzoic acid (DHB), sodium borohydride (NaBH4), (glucose, xylose, sucrose, maltose, raffinose, ribose, lactose, trechalose, and galactose), (cytosine, cytidine, and uridine), glutathione (GSH), triolein and (oleic acid, linoleic acid, linolenic acid, palmitic acid, and arachidonic acid) were purchased from Sigma-Aldrich (St, Louis, MO, USA). Atropine, carbamazepine, azedarachol, gastrodin and 3-O-tigloylswietenolide were obtained from Aladdin (Shanghai, China). (W-G, G-W-G, W-L, F-A,

F-A-N, F-G-G, F-E and Y-V) were obtained from SERVA Electrophoresis GmbH (Heidelberg, Germany). 1- propargyl-O-maltose was synthesized according to previous report.1 Acetonitrile (ACN) was purchased from

Merck (Darmstadt, Germany). Chloroauric acid (HAuCl4) was purchased from Shenyang Nonferrous Metal

Research Institute (Shenyang, China). Ammonium bicarbonate (NH4HCO3), sodium hydroxide (NaOH), concentration hydrochloric acid (HCl), nitric acid, anhydrous ethanol and methanol were obtained from Tianjin

Kermel plant of chemical reagent (Tianjin, China). Pure was prepared with a Milli-Q system (Millpore,

Bedford, MA, USA).

Synthesis of Au NCs

All of the glassware was cleaned with aqua regia before use. Au NCs were prepared according to previous

2,3 reports. Typically, an aqueous solution of HAuCl4 (40 mM, 3.75 mL) was added into ultrapure water (141.75 mL), followed by the addition of an aqueous Cys solution (50 mM, 4.5 mL) and the pH of the mixture was adjusted to approximately 12 by using a NaOH solution (1 M). Next, the reaction solution was reduced by using NaBH4 (57 mg, 1.5 mmol) for 3 h. Finally, the Au NCs were precipitated by the addition of ethanol (to 60%) and re-dissolved in ultrapure water for subsequent usage.

Synthesis of Au NPs

The Au NPs were synthesized as previous report.4 Aqueous solution of trisodium citrate (50 mL, 4 mM) was heated to boil under reflux with stirring. HAuCl4 solution (0.25 mL, 200 mM) was added into the boiled solution.

After heat for 3 min with the color change from pale yellow to wine-red, the Au NPs were obtained.

Materials Characterization

Transmission electron microscopy (TEM) images were recorded on a JEOL JEM-2000 EX TEM system

(JEOL, Tokyo, Japan). Ultraviolet–visible light (UV–vis) absorption spectra were recorded on a UV–Vis spectrophotometer (UV5, METTLER TOLEDO, Shanghai, China) in a wavelength range of 200-900 nm.

S-1 Thermogravimetric analysis (TGA) was performed under a nitrogen atmosphere with a heating rate of 10 °C min−1 from room temperature to 800 °C (NETZSCH, Selb, Germany).

Sample Preparation for MALDI-MS

The stock solutions of sugars, nucleosides, , fatty acid, natural products, alkaline drugs and peptides were firstly prepared in the solvents at the concentration of 10 mM. The details of solvents for analytes preparation were listed in Table S1. Then, the samples with different concentration were prepared by step dilution. The solution of

DHB matrix was prepared by dissolving DHB in 1 mL H2O/ACN/H3PO4 (29:70:1, v/v/v) at the concentration of

25 mg/mL. Au NCs (2.5 mg) were dissolved in 1 mL water. Au NPs (10 mg) were dispersed in 1 mL water.

Au NCs or Au NPs (0.5 μL) was firstly pipetted onto the plate and dried under ambient condition. Then, analyte solution (0.5 μL) was dropped onto the steel plate for MALDI-MS detection. When DHB was utilized, solution of analyte (0.5 μL) was pipetted onto the plate and dried under ambient condition. Then DHB solution

(0.5 μL) was dropped onto the steel plate for MALDI-MS detection.

All MALDI-MS detection were carried out on an AB SCIEX TOF/TOF 5800 (AB, SCIEX, USA) equipped with a pulsed Nd/YAG laser at 355 nm in positive reflective modes. All spectra were obtained by averaging 1000 laser shots. The laser irradiation point was automatically changed every 40 laser pulses, using the raster scan mode.

The reproducibility experiment was processed as follow: every analyte was analyzed on MALDI-TOF MS

(AB Sciex 5800) for ten times and the analysis area of analyte spot was randomly selected without any manual selection.

Mass spectrometry imaging.

The wild-type male Kunming mouse was provided by Dalian Medical University. The mice were anesthetized with carbon dioxide followed by cervical dislocation. Organs were harvested within 15 minutes. The animal experiments were processed according to the NIH Guide for the Care and Use of Laboratory Animals (National

Institutes of Health Publication, No.3040-2, revised 1999, Bethesda, MD) and were approved by the Animal Care and Use Committee of the Chinese Academy of Sciences. The fresh tissue was frozen quickly and cryosectioned by a Leica CM1950 cryostat (Leica Biosystems, Nussloch, Germany) to form 10 μm thickness sections. The tissue cryosections were then transferred onto a ITO glass slide (Bruker Daltonics, Bremen, Germany) and were desiccated in vacuum for 60 minutes at room temperature. The Au NCs solution of 2.5 mg/mL in water was dropped on the tissue slices by pipettor. In detail, Au NCs were dissolved in water with the concentration of 2.5 mg/mL.

Then Au NCs solution was dropped on the tissue slices with a pipettor (10 μL/drop). Finally, a homogenous

S-2 solution layer was formed onto the tissue slice. The MSI were performed after water evaporation. DHB (30 mg/mL,

50% methanol and 2% TFA) was sprayed by the Bruker Image Prep device (Bruker Daltonics, Bremen, Germany) with the default parameters.

MSI were processed on a Solarix 15T AS FTICR MS (Bruker Daltonics, Bremen, Germany) equipped with an MALDI source. The imaging data was acquired in positive ion mode with laser spot size setting at 50 μm, step width 100 μm and summed up by 200 shots at a laser repetition rate of 200 Hz. Imaging data were processed by

FlexImaging v4.0.

(1) Li, J.; Wang, F.; Liu, J.; Xiong, Z.; Huang, G.; Wan, H.; Liu, Z.; Cheng, K.; Zou, H. Chem. Commun. 2015,51, 4093-4096. (2) Tan, Y.; Liu, X. Y.; Zhang, L.; Wang, A.; Li, L.; Pan, X.; Miao, S.; Haruta, M.; Wei, H.; Wang, H.; Wang, F.; Wang, X.; Zhang, T. Angew. Chem. Int. Ed. 2017, 56, 2709-2713. (3) Li, J.; Liu, J.; Liu, Z.; Tan, Y.; Liu, X.; Wang, F. Anal. Chem. 2017, 89, 4339-4343. (4) Xu, G.; Liu, S.; Peng, J.; Lv, W.; Wu, R. ACS applied materials & interfaces 2015, 7, 2032-2038.

S-3 2. Supporting Fig.s and Tables

Fig. S1 (A) Illustration of the synthesis of homogeneous Au NCs; (B) Typical TEM image of Au NCs (Left) and

Au NPs (Right) with scale bar of 20 nm; (C) TGA results of Au NCs under N2 atmosphere with increasing temperature at 10 °C min-1; (D) UV−vis absorption spectrum for as-prepared Au NCs and Au NPs.

S-4 Fig. S2 Optical images of DHB crystal (A), DHB-analyte cocrystal (B), Au NCs (C), Au NCs-analyte cocrystal (D), Au NPs (E), and Au NPs-analyte cocrystal (F) on the MALDI steel plate.

S-5 Fig. S3 The MS spectra of glucose with the different concentration of Au NCs as matrix.

S-6 Fig. S4 Mass spectra of carbamazepine (A, m/z = 237.9 [M+H]+, 258.8 [M+Na]+, 274.8 [M+K]+ and 280.8 [M+2Na-H]+), cytosine (B, m/z = 111.9 [M+H]+, 133.9 [M+Na]+, 149.9 [M+K]+ and 155.9 [M+2Na-H]+), glutathione (C, m/z = 307.9 [M+H]+ and 329.9 [M+Na]+), and trechalose (D, m/z = 364.9 [M+Na]+) with Au NCs (a), DHB (b), Au NPs (c) as matrix, or without any matrix (d) for MALDI-MS detection in the positive-ion reflector mode. The amount of analyte was 5 nmol.

S-7 Fig. S5 Mass spectra of xylose (A, m/z = 172.8 [M+Na]+), trechalose (B, m/z = 364.9 [M+Na]+ and 386.9 [M+2Na- H]+), sucrose (C, m/z = 364.9 [M+Na]+, 380.9 [M+K]+ and 387.0 [M+2Na-H]+ ), ribose (D, m/z = 172.9 [M+Na]+), raffinose (E, m/z = 527.0 [M+Na]+), maltose (F, m/z = 364.9 [M+Na]+), lactose (G, m/z = 364.9 [M+Na]+ and 381.0 [M+K]+), glucose (H, m/z = 202.9 [M+Na]+), galactose (I, m/z = 202.9 [M+Na]+) and 1-progargyl-O-maltose (J, m/z = 402.8 [M+Na]+) with Au NCs for MALDI-MS detection in the positive-ion reflector mode. The amount of analyte was 5 nmol.

S-8 Fig. S6 Mass spectra of cytidine (A, m/z = 243.9 [M+H]+,265.9 [M+Na]+ and 281.9 [M+K]+), cytosine (B, m/z = 111.0 [M+H]+,133.9 [M+Na]+, 149.9 [M+K]+ and 155.9 [M+2Na-H]+), uridine (C, m/z = 266.9 [M+Na]+), triolein (D, m/z = 907.8 [M+Na]+), oleic acid (E, m/z = 305.4 [M+Na]+ and 327.4 [M+2Na-H]+), linoleic acid (F, m/z = 303.8 [M+Na]+ and 325.4 [M+2Na-H]+), linolenic acid (G, m/z = 301.4 [M+Na]+, 323.5 [M+2Na-H]+ and 339.4 [M+Na+K-H]+), palmitic acid (H, m/z = 279.5 [M+Na]+ and 301.5 [M+2Na-H]+) and arachidonic acid (I, m/z = 327.3 [M+Na]+, 349.6 [M+2Na-H]+ and 365.4 [M+Na+K-H]+) with Au NCs for MALDI-MS detection in the positive-ion reflector mode. The amount of analyte was 5 nmol.

S-9 Fig. S7 Mass spectra of 3-O-tigloylswietenolide (A, m/z = 591.0 [M+Na]+), atropine (B, m/z =290.0 [M+H]+, 311.9 [M+Na]+ and 327.9 [M+K]+), azedarachol (C, m/z = 443.0 [M+Na]+), gastrodin (D, m/z = 309.0 [M+Na]+, 325.0 [M+K]+ and 331.0 [M+2Na-H]+) and carbamazepine (E, m/z = 258.8 [M+Na]+, 274.8 [M+K]+ and 250.8 [M+2Na- H]+) with Au NCs for MALDI-MS detection in the positive-ion reflector mode. The amount of analyte was 5 nmol.

S-10 Fig. S8 Mass spectra of W-G (A, m/z = 261.9 [M+H]+ and 283.9 [M+Na]+), G-W-G (B, m/z = 318.9 [M+H]+ and 341.0 [M+Na]+), W-L (C, m/z = 318.0 [M+H]+ and 340.1 [M+Na]+ ), F-A (D, m/z = 236.9 [M+H]+ and 250.9 [M+Na]+), F-A-N (E, m/z = 351.0 [M+H]+ and 373.1 [M+Na]+), F-G-G (F, m/z = 280.0 [M+H]+ and 302.0 [M+Na]+), F-E (G, m/z = 295.0 [M+H]+ and 317.0 [M+Na]+), YV (H, m/z = 281.0 [M+H]+ and 303.1 [M+Na]+) and glutathione (I, m/z = 308.0 [M+H]+ and 330.0 [M+Na]+) with Au NCs for MALDI-MS detection in the positive-ion reflector mode. The amount of analyte was 5 nmol.

S-11 Fig. S9 Heatmap generated from the relative deviation of MS intensity related to the analytes commonly detected with Au NCs and DHB as matrix. Relative deviation is the difference between 1 and the ratio of MS intensity experimental value and average value

S-12 Fig. S10 Mass spectra of 1-propargyl-O-maltose with Au NCs as matrix. The analyte was 5 nmol with different concentration of NaCl (i), NH4HCO3 (ii) and

BSA (iii). The solvent is H2O, H2O and 10 mM NH4HCO3, respectively.

Fig. S11 Mass spectra of peptide F-E with Au NCs as matrix. The analyte was 5 nmol with different concentration of NaCl (i), NH4HCO3 (ii), and BSA (iii).

The solvent is H2O, H2O, and 10 mM NH4HCO3, respectively.

S-13 Fig. S12 Mass spectra of cytosine with Au NCs as matrix. The analyte was prepared in the amount of 5 nmol with different concentration of NaCl (i), NH4HCO3

(ii), and BSA (iii). The solvent is H2O, H2O, and 10 mM NH4HCO3, respectively.

S-14 Fig. S13 Average MS spectra of Au NCs-assisted MSI of mouse brain slice.

S-15 Fig. S14 Average MS spectra of DHB-assisted MSI of mouse brain slice. The peaks signed with × were caused by DHB.

S-16 Fig. S15 MS images related to PAs, PCs and other kinds of biomolecules commonly detected by both Au NCs and DHB assisted MSI.

S-17 Fig. S16 MS images related to biomolecules detected only by Au NCs assisted MSI.

S-18 Fig. S17 MS images related to biomolecules detected only by DHB assisted MSI.

S-19 Table S1 The chemical structure, special charge of relative ions, detection RSD (Au NCs and DHB), and LODs (Au NCs) of analytes. RSD (%) LODs Analyte Chemical structure Molecular weight solvent [M+H]+ [M+Na]+ [M+K]+ [M+2Na-H]+ [M+Na+K-H]+ Au NCs DHB (pmol)

HO O

xylose 150.15 H2O - 172.8 - - - 5.6 - 50 HO OH

OH

OH OH

O OH

trechalose 338.35 H2O - 364.9 - 386.9 - 6.5 - 5

HO OH OH

OH HO

OH OH

HO O OH

sucrose O O 342.30 H2O - 364.9 380.9 387.0 - 7.5 - 5 HO OH

OH HO OH

O OH

ribose 150.15 H2O - 170.0 - - - 3.0 - 5

HO OH

OH

HO OH OH

O OH HO O O raffinose 504.44 H2O - 547.0 - - - 4.3 - 5 O O OH

HO

OH OH OH

S-20 OH OH

HO O OH

maltose O 342.30 H2O - 364.9 - - - 5.0 - 5 HO O OH

OH HO

OH

HO OH

O lactose HO OH 342.30 H2O - 364.9 381.0 - - 6.5 - 5

O OH

HO O OH

HO OH HO

glucose 180.16 H2O - 202.9 - - - 4.3 - 5 O

HO OH

OH OH

O HO galactose 180.16 H2O - 202.9 - - - 3.8 - 5

OH OH

OH OH

HO O OH

O 1-propargyl-O-maltose HO O O 380.34 H2O - 402.8 - - - 7.4 - 5

OH HO

S-21 O

O O

O O O 3-O-tigloylswietenolide HO 568.65 50% ACN - 591.0 - - - 4.8 - 5

O O

CH3

atropine 288.38 ACN 290.0 311.9 327.9 - - 4.0 34.4 5

O OH

O

H

O

azedarachol O 420.58 75% ACN - 443.0 - - - 4.7 - 5 HO OH

HO

O O HO

gastrodin OH 286.28 50% ACN - 309.0 325.0 331.0 - 3.5 - 5 HO OH

OH

carbamazepine N 236.27 ACN 237.0 258.8 274.8 280.8 - 6.0 53.1 5

O NH2

S-22 OH HO OH cytidine 243.22 H2O 243.9 265.9 281.9 - - 12.2 55.6 5 N O

H2N N O

O N NH2 cytosine 111.10 H2O 111.0 133.9 149.9 155.9 - 5.7 26.8 5 HN

OH HO OH

uridine 244.20 H2O - 266.9 - - - 7.0 - 5 N O

O N O H

triolein 885.45 ethanol - 907.8 - - - 10.6 - 5

10 mM Oleic acid 282.46 - 305.4 - 327.4 - 7.1 - 5 NaOH

S-23 10 mM Linoleic acid 280.45 - 303.8 - 325.4 - 6.0 - 5 NaOH

10 mM Linolenic acid 278.43 - 301.4 323.5 339.4 5.2 - 5 NaOH

10 mM Palmitic acid 256.42 - 279.5 - 301.5 - 7.1 - 5 NaOH

10 mM Arachidonic acid 304.47 327.3 - 349.6 365.4 4.0 - 5 NaOH

O O

H H2N CH C N CH2 C OH

CH2

WG 261.28 H2O 261.9 283.9 - - 5.6 41.4 5

HN

S-24 O O O

H H H2N CH2 C N CH C N CH2 C OH

CH2

GWG 318.33 H2O 318.9 241.0 - - 6.9 36.5 5

HN

O O

H H2N CH C N CH C OH

CH2 CH2

CH CH3 WL 317.39 H2O 318.0 340.1 - - 7.5 44.4 5

CH3 HN

O O

H H2N CH C N CH C OH

CH2 CH3 FA 236.27 H2O 236.9 259.0 - - 8.3 25.2 5

O O O

H H H2N CH C N CH C N CH C OH

CH2 CH3 CH2

C O FAN 350.37 H2O 351.0 373.1 - - 6.9 34.1 5

NH2

S-25 O O O

H H H2N CH C N CH2 C N CH2 C OH

CH2 FGG 279.3 H2O 280.0 302.0 - - 6.5 22.1 5

O O

H H2N CH C N CH C OH

CH2 CH2 FE 294.31 H2O 295.0 317.0 - - 3.1 26.4 5 CH2

C O

OH

O O

H H2N CH C N CH C OH

CH2 CH CH3

CH3 YV 280.32 H2O 281.0 303.1 - - 4.4 29.0 5

OH

HS

O O O H GSH N 307.32 H2O 308.0 330.0 - - 6.4 11.6 5 HO N OH H

O NH2

S-26 Table S2 MS peak intensities and signal to noise ratios (S/N) of the seven kinds of biomolecules with Au NCs, DHB, and Au NPs as matrix, respectively. DHB Au NCs Au NPs ion m/z Analyte m/z ion Intensity S/N Intensity S/N Intensity S/N - - [M+H]+ 181.2 glucose 202.9 [M+Na]+ 7056 1133.43 750 42.78 4345 764.06 [M+H]+ 236.9 F-A 259.0 [M+Na]+ 14240 2416.21 10883 639.09 10848 1885.09 [M+H]+ 290.0 atropine 311.9 [M+Na]+ 9355 1321.82 843 49.25 - - [M+H]+ 343.3 lactose 364.9 [M+Na]+ 18311 2378.97 2653 209.12 - - [M+H]+ 421.3 azedarachol 443.0 [M+Na]+ 59430 7444.70 5870 464.71 - - [M+H]+ 569.7 3-O-tigloylswietenolide 591.0 [M+Na]+ 39102 5129.24 613 48.03 - - [M+H]+ 886.5 triolein 907.8 [M+Na]+ 43809 8024.53 292 22.97

S-27 Table S3 List of biomolecules commonly detected in MSI of mouse brain by both Au NCs and DHB. The * represents that the ion was recorded with high- quality MS images (with clear lateral distribution outlines). Matrix m/z Intensity Res. S/N Tentatively proposed identity Ions Class DHB 551.5373 1273004 77675 583.2 12:0Cholesterylester [M+H-H2O]+ CL Au NCs 605.55205 41420 75717 16.4 12:0Cholesterylester [M+K]+ CL DHB 230.95959 6204347 183412 1163.9 2,3-Bisphospho-D-glycerate [M+H-2H2O]+ Lipid Au NCs 288.81166 9286 112247 40.8 2,3-Bisphospho-D-glycerate [M+Na]+ Lipid * Au NCs 850.68343 385266 42007 2457.6 GalCer(d18:1/C24:0) [M+K]+ GalCer * DHB 850.70637 1054154 39194 404.2 GalCer(d18:1/C24:0) [M+K]+ GalCer * DHB 790.53498 648869 52522 126.7 GalCer(d38:3) [M+K]+ GalCer Au NCs 790.57419 1892428 42755 6.7 GalCer(d38:3) [M+K]+ GalCer * Au NCs 774.59907 129795 41822 5.2 GalCer(d38:3) [M+Na]+ GalCer DHB 832.63393 3472432 48290 316.3 GlcCer(d42:2) [M+Na]+ GlcCer Au NCs 832.72759 6151171 33515 10.4 GlcCer(d42:2) [M+Na]+ GlcCer * DHB 848.60743 5185344 45566 872.1 GlcCer(d42:2) [M+K]+ GlcCer * DHB 810.64925 9766736 50797 229.6 GlcCer(d42:2) [M+H]+ GlcCer Au NCs 848.701 20563890 33205 35.8 GlcCer(d42:2) [M+K]+ GlcCer DHB 697.52011 2090136 59429 198.2 PA(34:1) [M+Na]+ PA Au NCs 697.52936 732740 57399 129.3 PA(34:1) [M+Na]+ PA * DHB 713.49464 5593004 62428 66.7 PA(34:1) [M+K]+ PA Au NCs 713.50214 5682225 49402 738.2 PA(34:1) [M+K]+ PA DHB 725.55336 496914 61216 21.1 PA(36:1) [M+Na]+ PA Au NCs 725.56374 197709 57509 4.5 PA(36:1) [M+Na]+ PA * DHB 741.52812 1475097 58166 124.2 PA(36:1) [M+K]+ PA Au NCs 741.53654 1444131 55263 27.1 PA(36:1) [M+K]+ PA

S-28 DHB 723.53645 3326484 56465 279.2 PA(36:2) [M+Na]+ PA Au NCs 723.54264 2008441 51867 238 PA(36:2) [M+Na]+ PA DHB 739.51245 8346186 56645 121 PA(36:2) [M+K]+ PA Au NCs 739.52319 10545403 49289 1292.6 PA(36:2) [M+K]+ PA * DHB 767.54495 3053479 55422 36.8 PA(38:2) [M+K]+ PA * Au NCs 767.55393 3327161 47724 90.3 PA(38:2) [M+K]+ PA * DHB 763.51427 271746 53538 442.3 PA(38:4) [M+K]+ PA * Au NCs 763.52451 638758 46685 51.6 PA(38:4) [M+K]+ PA * DHB 773.5756 10365498 55723 25.6 PA(40:5) [M+Na]+ PA Au NCs 773.58687 1884056 51285 8.2 PA(40:5) [M+Na]+ PA * Au NCs 789.45198 1489558 32636 431 PA(40:5) [M+K]+ PA DHB 813.53288 1245057 49234 59.9 PA(42:7) [M+K]+ PA Au NCs 813.54885 913102 49016 20.7 PA(42:7) [M+K]+ PA DHB 726.55687 101213 55166 275.3 PC(30:1) [M+Na]+ PC Au NCs 726.56668 51742 57800 7.3 PC(30:1) [M+Na]+ PC DHB 724.54071 1326155 52210 104.5 PC(30:2) [M+Na]+ PC Au NCs 724.54975 605325 44645 21.6 PC(30:2) [M+Na]+ PC Au NCs 740.52757 3280175 55770 76.1 PC(30:2) [M+K]+ PC * DHB 697.52011 2090136 59429 198.2 PC(32:0) [M-N(CH3)3+Na]+ PC Au NCs 697.52936 732740 57399 129.3 PC(32:0) [M-N(CH3)3+Na]+ PC DHB 772.57231 24086466 54137 52.3 PC(32:0) [M+K]+ PC Au NCs 772.58559 5523139 53864 7 PC(32:0) [M+K]+ PC Au NCs 713.50214 5682225 49402 738.2 PC(32:0) [M-N(CH3)3+K]+ PC * DHB 734.61348 19405044 58240 27.2 PC(32:0) [M+H]+ PC * Au NCs 756.60629 1909012 50874 26.1 PC(32:0) [M+Na]+ PC

S-29 DHB 770.55576 1396913 52947 435.6 PC(32:1) [M+K]+ PC * Au NCs 770.56394 469232 53075 28 PC(32:1) [M+K]+ PC * DHB 732.59787 1052277 56196 281.5 PC(32:1) [M+H]+ PC DHB 768.54787 1316000 53027 132.4 PC(32:2) [M+K]+ PC * Au NCs 768.55472 1283662 50404 156.1 PC(32:2) [M+K]+ PC * DHB 725.55336 496914 61216 21.1 PC(34:0) [M-N(CH3)3+Na]+ PC Au NCs 725.56374 197709 57509 4.5 PC(34:0) [M-N(CH3)3+Na]+ PC DHB 784.57251 1118605 55755 238.6 PC(34:0) [M+Na]+ PC Au NCs 784.64129 382398 44457 577 PC(34:0) [M+Na]+ PC DHB 800.60781 5872647 53070 53.9 PC(34:0) [M+K]+ PC Au NCs 800.62109 1202607 41542 248.8 PC(34:0) [M+K]+ PC * DHB 762.64779 5174029 57931 17.4 PC(34:0) [M+H]+ PC * Au NCs 798.59723 13629953 39629 22.3 PC(34:1) [M+K]+ PC Au NCs 723.54264 2008441 51867 238 PC(34:1) [M-N(CH3)3+Na]+ PC Au NCs 739.52319 10545403 49289 1292.6 PC(34:1) [M-N(CH3)3+K]+ PC DHB 760.63006 31313948 54574 87.7 PC(34:1) [M+H]+ PC * Au NCs 782.62905 3171802 42477 43.1 PC(34:1) [M+Na]+ PC * DHB 756.59679 8053693 52542 591.8 PC(34:3) [M+H]+ PC Au NCs 794.50739 710518 46248 26 PC(34:3) [M+K]+ PC * Au NCs 814.57347 3397692 47553 26.1 PC(35:0) [M+K]+ PC * DHB 814.58452 1772988 49609 65.2 PC(35:0) [M+K]+ PC DHB 828.60233 3055295 61308 177.5 PC(36:0) [M+K]+ PC Au NCs 828.61565 297044 38427 824.6 PC(36:0) [M+K]+ PC * DHB 826.62322 13363591 48174 1199.3 PC(36:1) [M+K]+ PC * Au NCs 826.63752 2469512 47862 34.4 PC(36:1) [M+K]+ PC

S-30 Au NCs 767.55393 3327161 47724 90.3 PC(36:1) [M-N(CH3)3+K]+ PC * Au NCs 810.6634 617923 50850 4.9 PC(36:1) [M+Na]+ PC DHB 784.6322 2546796 49260 24.7 PC(36:3) [M+H]+ PC Au NCs 822.54518 7209155 45555 3045.1 PC(36:3) [M+K]+ PC * Au NCs 761.50552 1865660 47015 186.1 PC(36:4) [M-N(CH3)3+K]+ PC * DHB 782.6154 19914362 52562 123.9 PC(36:4) [M+H]+ PC Au NCs 820.52616 1226673 40758 552.5 PC(36:4) [M+K]+ PC DHB 820.57536 5537440 49330 72.2 PC(36:4) [M+Na]+ PC DHB 802.61404 440859 52561 149.2 PC(36:5) [M+Na]+ PC Au NCs 818.56338 264999 37444 260.1 PC(36:5) [M+K]+ PC * Au NCs 856.58556 1466084 45124 46.7 PC(38:0)U [M+K]+ PC DHB 856.63486 1531533 46886 282.4 PC(38:0)U [M+K]+ PC DHB 834.65146 4481193 56061 88 PC(38:3) [M+Na]+ PC Au NCs 834.74463 1410136 36342 8.1 PC(38:3) [M+Na]+ PC DHB 812.65309 976565 49366 54.8 PC(38:3) [M+H]+ PC Au NCs 850.71567 4942570 38052 4505.8 PC(38:3) [M+K]+ PC * DHB 832.63393 3472432 48290 316.3 PC(38:4) [M+Na]+ PC * Au NCs 832.72759 6151171 33515 10.4 PC(38:4) [M+Na]+ PC * DHB 848.60743 5185344 45566 872.1 PC(38:4) [M+K]+ PC Au NCs 848.701 20563890 33205 35.8 PC(38:4) [M+K]+ PC DHB 810.64925 9766736 50797 229.6 PC(38:4) [M+H]+ PC DHB 846.59455 1116581 47953 30.9 PC(38:5) [M+K]+ PC Au NCs 846.6037 104006 37333 301.7 PC(38:5) [M+K]+ PC * Au NCs 844.5326 6359750 41747 438.6 PC(38:6) [M+K]+ PC DHB 844.57673 7384906 48748 61.4 PC(38:6) [M+K]+ PC

S-31 Au NCs 785.50826 1825245 46216 197.5 PC(38:6) [M-N(CH3)3+K]+ PC * DHB 806.61811 7979621 50064 89 PC(38:6) [M+H]+ PC * DHB 804.59982 2345520 50641 197.6 PC(38:7) [M+H]+ PC Au NCs 842.51752 833295 34161 35.4 PC(38:7) [M+K]+ PC * Au NCs 872.55615 1028838 43793 171.8 PC(40:6) [M+K]+ PC * DHB 872.60855 3713172 47231 596.6 PC(40:6) [M+K]+ PC DHB 896.52837 238252 44108 7867 PC(41:1) [M+K]+ PC * Au NCs 896.54627 389720 32539 33 PC(41:1) [M+K]+ PC DHB 788.66392 12540497 51188 361.1 PC(O-37:1) [M+H]+ PC * Au NCs 826.63752 2469512 47862 34.4 PC(O-37:1) [M+K]+ PC * DHB 822.69331 1889111 49175 163.2 PC(O-38:2) [M+Na]+ PC Au NCs 822.70478 8177602 40777 139.4 PC(O-38:2) [M+Na]+ PC * DHB 838.66821 4903644 46175 515.5 PC(O-38:2) [M+K]+ PC * Au NCs 838.68005 26509794 39995 39.6 PC(O-38:2) [M+K]+ PC DHB 866.70142 4635932 34512 30.2 PC(O-40:2) [M+K]+ PC * Au NCs 866.71364 29235572 38449 320.8 PC(O-40:2) [M+K]+ PC * Au NCs 850.73319 9061546 34902 268 PC(O-40:2) [M+Na]+ PC DHB 806.55826 2824550 49223 1508.3 PC(P-36:3) [M+K]+ PC * Au NCs 806.5679 10077244 43321 173 PC(P-36:3) [M+K]+ PC * DHB 830.56029 7359352 32591 173.9 PC(P-38:5) [M+K]+ PC * Au NCs 830.57294 10798352 36957 111.1 PC(P-38:5) [M+K]+ PC * DHB 770.61153 2519120 53575 39 PE(36:0) [M+Na]+ PE Au NCs 770.62004 89526 46104 27.8 PE(36:0) [M+Na]+ PE DHB 748.63047 1324094 59276 30.3 PE(36:0) [M+H]+ PE * Au NCs 786.54271 1029923 53676 22.6 PE(36:0) [M+K]+ PE *

S-32 DHB 766.53314 213899 55814 32.9 PE(36:2) [M+Na]+ PE * Au NCs 766.57129 528812 43843 242.9 PE(36:2) [M+Na]+ PE * DHB 740.51627 3439617 60836 54.1 PE(36:4) [M+H]+ PE Au NCs 762.53858 561945 49248 26 PE(36:4) [M+Na]+ PE * Au NCs 778.53328 773980 37439 229.6 PE(36:4) [M+K]+ PE DHB 774.64806 2605779 44762 23.3 PE(38:1) [M+H]+ PE Au NCs 796.58267 679187 45113 35.7 PE(38:1) [M+Na]+ PE Au NCs 812.55741 2258508 48488 158.5 PE(38:1) [M+K]+ PE * DHB 806.55826 2824550 49223 1508.3 PE(38:4) [M+K]+ PE * Au NCs 806.5679 10077244 43321 173 PE(38:4) [M+K]+ PE * DHB 802.52676 1542300 65633 83.3 PE(38:6) [M+K]+ PE Au NCs 802.5369 1264590 53255 7.9 PE(38:6) [M+K]+ PE * DHB 796.63276 553535 53642 28 PE(40:4) [M+H]+ PE Au NCs 818.60302 699150 48022 9.7 PE(40:4) [M+Na]+ PE * Au NCs 834.59944 1617624 26956 39.4 PE(40:4) [M+K]+ PE DHB 792.60156 2130360 54508 88.3 PE(40:6) [M+H]+ PE Au NCs 814.57347 3397692 47553 26.1 PE(40:6) [M+Na]+ PE * Au NCs 830.57294 10798352 36957 111.1 PE(40:6) [M+K]+ PE DHB 810.50284 129892 48237 1846.1 PE(40:8) [M+Na]+ PE * Au NCs 810.51513 471272 39444 291.7 PE(40:8) [M+Na]+ PE Au NCs 826.51616 834281 37220 256.5 PE(40:8) [M+K]+ PE * DHB 766.53314 213899 55814 32.9 PE(O-36:3) [M+K]+ PE * Au NCs 766.61684 769020 45076 4.7 PE(O-36:3) [M+K]+ PE * Au NCs 750.59287 19922 52169 71.6 PE(O-36:3) [M+Na]+ PE DHB 758.61345 1438909 52328 101.8 PE(O-38:2) [M+H]+ PE

S-33 DHB 780.5986 779384 53809 23.5 PE(O-38:2) [M+Na]+ PE Au NCs 796.61939 96651 47710 15.4 PE(O-38:2) [M+K]+ PE * DHB 754.58062 379925 55758 194.7 PE(O-38:4) [M+H]+ PE Au NCs 792.54939 281141 52570 14.8 PE(O-38:4) [M+K]+ PE * DHB 774.58139 1774483 51139 450.6 PE(O-38:5) [M+Na]+ PE Au NCs 774.59907 129795 41822 5.2 PE(O-38:5) [M+Na]+ PE DHB 790.53498 648869 52522 126.7 PE(O-38:5) [M+K]+ PE DHB 752.57391 329629 58112 44 PE(p-38:4) [M+H]+ PE Au NCs 790.54779 371864 31805 598.3 PE(p-38:4) [M+K]+ PE * Au NCs 845.52983 2261217 45341 165.5 PG(40:6) [M+Na]+ PG * DHB 845.57932 3604488 52061 65.3 PG(40:6) [M+Na]+ PG Au NCs 834.54017 1206105 39411 804.1 PS(38:4) [M+Na]+ PS DHB 834.59124 425305 48713 986.6 PS(38:4) [M+Na]+ PS Au NCs 850.5727 1460427 37130 191.4 PS(38:4) [M+K]+ PS * DHB 753.63386 2239413 54871 56.9 TG(43:3) [M+Na]+ TG DHB 769.60822 5535704 52267 275.8 TG(43:3) [M+K]+ TG Au NCs 769.6169 238467 48808 27 TG(43:3) [M+K]+ TG DHB 731.6503 3131533 56012 221.5 TG(43:3) [M+H]+ TG

S-34 Table S4 List of analytes detected in MSI of mouse brain by Au NCs. The * represents that the ion was recorded with high-quality MS images (with clear lateral distribution outlines). m/z Intensity Res. S/N Tentatively proposed identity Ions Class 295.07549 6666 115607 1066.4 5-S-Cysteinyldopamine [M+Na]+ Alkaloid 588.5754 39294 72005 33.1 Cer(d36:1) [M+Na]+ Cer 604.54982 139297 66986 19.9 Cer(d36:1) [M+K]+ Cer 425.34634 49951 85794 438.3 [M+K]+ CL * 457.31388 30221 94923 19.1 CPA(18:1) [M+K]+ PA 711.54659 91252 55973 12.4 DAG(41:3) [M+Na]+ DAG 367.22721 23006 99607 58.9 Docosahexaenoic acid [M+K]+ FA 279.00584 86670 126043 30.9 FA14:0 [M+K]+ FA * 710.53917 254032 44032 67.8 GalCer(d18:1/C14:0) [M+K]+ GalCer 750.63935 182816 54072 4.9 GalCer(d18:1/C18:0) [M+Na]+ GalCer 820.67357 504660 44844 56.7 GalCer(d18:1/C22:1) [M+K]+ GalCer * 846.68389 607140 36059 393.4 GalCer(d18:1/C24:2) [M+K]+ GalCer * 810.54413 783867 50392 15.8 GlcCer(d38:1) [M+K]+ GlcCer * 457.24302 6215 81612 162.8 MetLeuAlaThr [M+Na]+ Peptides 415.2886 191538 87610 84.6 PA(16:0) [M+Na]+ PA 701.50158 54248 44716 30.5 PA(33:0) [M+K]+ PA 765.48033 640936 44457 186.2 PA(38:3) [M+K]+ PA * 761.50552 1865660 47015 186.1 PA(38:5) [M+K]+ PA * 791.57364 762986 47504 88.7 PA(40:4) [M+K]+ PA * 787.5249 669729 42844 181.6 PA(40:6) [M+K]+ PA * 771.4912 89058 33091 57.9 PA(40:6) [M+Na]+ PA 785.50826 1825245 46216 197.5 PA(40:7) [M+K]+ PA *

S-35 707.55218 245519 55622 4.4 PA(O-36:3) [M+Na]+ PA * 723.54264 2008441 51867 238 PA(O-36:3) [M+K]+ PA 433.35914 963165 78195 1763.6 PA(P-16:0) [M+K]+ PA * 784.58133 1617958 52300 120.7 PC(33:1) [M+K]+ PC * 796.52526 2655092 45151 252.6 PC(34:2) [M+K]+ PC * 788.52515 211543 46815 640.4 PC(34:6) [M+K]+ PC * 765.48033 640936 44457 186.2 PC(36:2) [M-N(CH3)3+K]+ PC 808.52655 1554988 50844 159.2 PC(36:2) [M+Na]+ PC * 824.61679 614917 55772 91 PC(36:2) [M+K]+ PC 836.55943 5160558 42153 525.5 PC(37:3) [M+K]+ PC * 834.59944 1617624 26956 39.4 PC(O-38:4) [M+K]+ PC * 852.73712 505746 47251 39.7 PC(O-40:1) [M+Na]+ PC * 864.69562 12470138 40125 4.3 PC(P-41:2) [M+K]+ PC * 728.52353 65016 49665 7 PE(32:1) [M+K]+ PE 710.53917 254032 44032 67.8 PE(32:2) [M+Na]+ PE 742.54067 535399 57549 14.7 PE(34:0) [M+K]+ PE 740.52757 3280175 55770 76.1 PE(34:1) [M+K]+ PE * 768.58316 494718 50870 52.3 PE(36:1) [M+Na]+ PE * 780.54942 2136239 51403 14.9 PE(36:3) [M+K]+ PE * 794.60586 206765 35476 128 PE(38:2) [M+Na]+ PE 810.60408 43136 39279 316.8 PE(38:2) [M+K]+ PE * 804.53122 1721037 39154 570.2 PE(38:3) [M+K]+ PE * 780.49928 152272 46309 675.4 PE(38:9) [M+Na]+ PE * 822.70478 8177602 40777 139.4 PE(40:2) [M+Na]+ PE 838.68005 26509794 39995 39.6 PE(40:2) [M+K]+ PE *

S-36 820.58724 1484320 37909 187.7 PE(40:3) [M+Na]+ PE * 828.55734 5909788 37840 110.4 PE(40:7) [M+K]+ PE * 824.50019 1726166 27856 789 PE(40:9) [M+Na]+ PE * 838.57489 620035 47382 13182.2 PE(42:8) [M+Na]+ PE * 854.55109 759443 47739 249.5 PE(42:8) [M+K]+ PE 836.72219 1663453 45882 72.2 PE(42:9) [M+Na]+ PE * 852.55672 22029218 45550 2938.3 PE(42:9) [M+K]+ PE * 884.52427 44586 36983 25.3 PE(44:7) [M+K]+ PE 740.55286 194499 68944 66.4 PE(O-34:2) [M+K]+ PE * 764.57747 259214 45319 202.4 PE(O-36:4) [M+K]+ PE * 782.62905 3171802 42477 43.1 PE(O-37:2) [M+K]+ PE * 782.62905 3171802 42477 43.1 PE(O-38:1) [M+Na]+ PE * 794.56644 181502 44875 76.7 PE(O-38:3) [M+K]+ PE * 836.66457 1057181 36368 826.9 PE(O-44:2) [M+Na]+ PE * 852.69456 5909283 37228 251.3 PE(O-44:2) [M+K]+ PE 786.54271 1029923 53676 22.6 PE(P-38:6) [M+K]+ PE * 794.67129 344465 38506 133.2 PE(P-39:1) [M+Na]+ PE 810.64636 851403 44648 229.9 PE(P-39:1) [M+K]+ PE * 814.54542 237764 38691 1265.1 PE(P-40:6) [M+K]+ PE * 697.52936 732740 57399 129.3 PE-Cer(d34:2) [M+K]+ Cer-PE * 787.5249 669729 42844 181.6 PG(34:1) [M+K]+ PG * 771.4912 89058 33091 57.9 PG(34:1) [M+Na]+ PG 779.49489 726890 36693 93.7 PG(34:5) [M+K]+ PG * 777.48402 1183135 44113 68 PG(34:6) [M+K]+ PG 758.56372 629310 54260 20.5 PS(32:0) [M+Na]+ PS *

S-37 782.57145 281095 44852 1002.9 PS(34:2) [M+Na]+ PS * 813.04828 15644 36617 17.6 PS(36:1) [M+Na]+ PS * 828.55734 5909788 37840 110.4 PS(36:1) [M+K]+ PS * 826.53397 689401 41642 919.4 PS(36:2) [M+K]+ PS * 822.54518 7209155 45555 3045.1 PS(36:4) [M+K]+ PS * 874.56326 620169 18604 140.2 PS(40:6) [M+K]+ PS 502.29718 229384 46373 63.1 Ser Phe Val Lys [M+Na]+ Peptides 753.64441 62910 54691 12.7 SM(d18:1/C18:0) [M+Na]+ SM 769.53138 360592 35818 15.3 SM(d18:1/C18:0) [M+K]+ SM

S-38 Table S5 List of analytes detected in MSI mouse brain by DHB. The * represents that the ion was recorded with high-quality MS images (with clear lateral distribution outlines). m/z Intensity Res. S/N compound Ions Class 577.55424 2155787 71739 1027.2 14:1Cholesterylester [M+H-H2O]+ CL 199.00987 561858 213303 1048.7 3-Phospho-D-erythronate [M+H-H2O]+ Lipid 329.02562 19033130 118795 9500.4 D-glycero-D-manno-Heptose1-phosphate [M+K]+ Lipid 754.63651 925101 52952 655.1 GalCer(d18:1/C20:1) [M+H]+ GalCer 810.73137 1214831 53475 55.6 GalCer(d18:1/C24:1) [M+H]+ GalCer * 710.53129 1235430 62020 66.8 GalCer(d18:1C14:0) [M+K]+ GalCer 776.60555 1303268 58623 38.6 PC(34:4) [M+Na]+ PC 784.61705 1380044 56996 440.4 PC(O-34:1) [M+K]+ PC 864.68455 2373247 48970 69.7 PC(O-40:3) [M+K]+ PC 826.62322 13363591 48174 1199.3 PC(P-37:0) [M+K]+ PC * 784.61705 1380044 56996 440.4 PE(O-40:3) [M+H]+ PE * 785.49899 2155078 55631 60.9 PG(34:2) [M+K]+ PG * 222.04266 749337 182998 2585.8 Phosphocholine [M+K]+ Lipid 848.68898 3960895 47403 266.3 PS(40:0) [M+H]+ PS 870.63848 20712982 53160 106 PS(40:0) [M+Na]+ PS * 369.37422 1596615 100947 1205 5beta-Cholestane-3alpha,12alpha-diol [M+H-2H2O]+ CL * 391.30984 944301 91805 906.2 (23S)-3α,7β,23-Trihydroxy-5β-cholan-24-oic Acid [M+H-H2O]+ CL 808.63468 2321306 51796 66.7 PC(P-37:1) [M+Na]+ PC

S-39