Abd0957 SM

advances.sciencemag.
org/cgi/content/full/7/5/eabd0957/DC1
Supplementary Materials for
Spatially resolved 3D metabolomic profiling in tissues
Shambavi Ganesh, Thomas Hu, Eric Woods, Mayar Allam, Shuangyi Cai,
Walter Henderson, Ahmet F. Coskun*
*Corresponding author. Email: ahmet.coskun@bme.gatech.edu
Published 27 January 2021, Sci. Adv. 7, eabd0957 (2021)

DOI: 10.1126/sciadv.abd0957
The PDF file includes:
Figs. S1 to S37
Tables S1 to S11
Legends for data files S1 and S2
Other Supplementary Material for this manuscript includes the following:
(available at advances.sciencemag.org/cgi/content/full/7/5/eabd0957/DC1)
Data files S1 and S2

Fig. S1. Metabolic 3D correlations unlabeled tonsil tissues. Heatmaps of metabolic correlations using different
aggregation of the image slices. (A) Heatmap using the correlation of all the image slices. (B) Heatmap using the
correlation of the mean of every 5 image slices. (C) Heatmap using the correlation of the mean of every 20 image
slices. (D) Heatmap using the correlation of the mean of every image slices.
Fig. S2. Optical imaging for identification of region-of-interest before the metabolic analysis. (A) Keyence
optical microscope is utilized as the main optical imaging platform for the unlabeled and labeled tonsil tissue
slices. (B) The images from the Keyence optical microscope are matched spatially with the optical images
obtained from the internal optical camera in the TOF-SIMS platform. The 2D reconstructed images of mass
channels are also compared with the Keyence optical microscope images to validate the matching regions. (C)
Once the ROIs between the Keyence optical imaging platform and the TOF-SIMS platform are matched, using
landmarks on the tissue slide, the TOF-SIMS data measurement process begins.
Fig. S3. Optical camera inside the TOF-SIMS device to perform alignment on spatially distinct regions of
tonsil tissues. The upper row shows the image before the measurements and the bottom row corresponds to the
image after the measurement for the datasets unlabeled, inside, outside, and the border of the GC of the tonsil
slice. The area of the field of view (FOV) captured in the entirety of each 2D image is 815 µm x 771 µm. The red
square in each 2D image indicates the region of analysis for each dataset and the area within the red square is
150-µm x 150-µm. It can be observed that due to the sputtering action of TOF-SIMS on the tonsil tissue, the
images before measurement vary slightly compared to the images captured after measurement.
Fig. S4. Metabolic 3D correlations inside the germinal center in tonsil tissues. Heatmaps of metabolic
correlations using different aggregation of the image slices. (A) Heatmap using the correlation of all the image
slices. (B) Heatmap using the correlation of the mean of every 5 image slices. (C) Heatmap using the correlation
of the mean of every 20 image slices. (D) Heatmap using the correlation of the mean of every image slices.
Fig. S5. Metabolic 3D correlations outside the germinal center in tonsil tissues. Heatmaps of metabolic
correlations using different aggregation of the image slices. (A) Heatmap using the correlation of all the image
slices. (B) Heatmap using the correlation of the mean of every 5 image slices. (C) Heatmap using the correlation
of the mean of every 20 image slices. (D) Heatmap using the correlation of the mean of every image slices.
Fig. S6. Metabolic 3D correlations at the border region of the germinal center in tonsil tissues. Heatmaps
of metabolic correlations using different aggregation of the image slices. (A) Heatmap using the correlation of all
the image slices. (B) Heatmap using the correlation of the mean of every 5 image slices. (C) Heatmap using the
correlation of the mean of every 20 image slices. (D) Heatmap using the correlation of the mean of every image
slices.
Fig. S7. Bar plot of selected mass-channels inside the germinal center in labeled tonsil tissues. The mean
pixel intensities across all 2D slices in log scale are displayed on the y-axis, and the selected mass-channel
numbers are displayed on the x-axis. Mass-channels 84, 86, and 91 are among some compounds which are
significant in the barplot.
Fig. S8. Bar plot of selected mass-channels outside the germinal center in labeled tonsil tissues. The mean
pixel intensities across all 2D slices in log scale are displayed on the y-axis, and the selected mass-channel
Fig. S9. Bar plot of selected mass-channels on the border the germinal center in labeled tonsil tissues. The
mean pixel intensities across all 2D slices in log scale are displayed on the y-axis, and the selected mass-channel
Fig. S10. Bar plot of selected mass-channels in unlabeled tonsil tissues. The mean pixel intensities across all
2D slices in log scale are displayed on the y-axis, and the selected mass-channel numbers are displayed on the x-
axis. Mass-channels 73, 74, and 185 are among some compounds which are significant in the barplot.
Fig. S11. Bar plot of comparison of isotope labels across inside, outside, and the border of the GC in labeled
tonsil datasets. The mean pixel intensities across all 2D slices in log scale are displayed on the y-axis, and the
isotope label numbers are displayed on the x-axis. Isotope labels of m/z 141, 143, and 156 are among some
compounds which are significant in the barplot for the inside of the germinal center labeled tonsil datasets. Isotope
labels of m/z 141, 156, and 159 are among some compounds which are significant in the barplot for outside of
the germinal center labeled tonsil datasets. Isotope labels of m/z 141, 156, and 159 are among some compounds
which are significant in the barplot for outside of the germinal center labeled tonsil datasets. Isotope labels of
m/z 156, 170, and 171 are among some compounds which are significant in the barplot for the border of the
germinal center labeled tonsil datasets. For the comparison of the 3 datasets, the isotope labels on the inside of
the labeled dataset are the most prominent, and the isotope labels on the outside of the labeled dataset are the least
prominent. The border of the germinal center dataset isotope labels are indicative of the transition state between
the inside and outside the germinal center labeled dataset.
Fig. S12. Metabolic 2D ion images in unlabeled tonsil tissues. Mass-channel numbers and original 2D
metabolite images obtained from the reconstruction of TOF-SIMS measurement data are shown for the unlabeled
tonsil dataset. The 2D images for the selected mass-channels shown are 150-µm x 150-µm in area, reconstructed
by using the Surfacelab version 6 software for the unlabeled tonsil tissue dataset. The 2D image corresponding to
the mass channels are 4-pixel (x-y) binned.
Fig. S13. Metabolic 2D ion images inside the germinal center in tonsil tissues. Mass-channel numbers, along
with the original 2D antibody labels and metabolites images obtained from reconstruction of TOF-SIMS
measurement data are shown for the inside of the germinal center tonsil dataset. The 2D images for the selected
mass-channels shown are 150-µm x 150-µm in area, reconstructed by using the Surfacelab version 6 software for
the inside of the germinal center tonsil tissue dataset. The 2D image corresponding to the mass channels are 4-
pixel (x-y) binned.
Fig. S14. Metabolic 2D ion images outside the germinal center in labeled tonsil tissues. Mass-channel
numbers, along with the original 2D antibody labels and metabolites images obtained from reconstruction of
TOF-SIMS measurement data are shown for the outside of the germinal center tonsil dataset. The 2D images for
the selected mass-channels shown are 150-µm x 150-µm in area, reconstructed by using the Surfacelab version 6
software for the outside of the germinal center tonsil tissue dataset. The 2D image corresponding to the mass
channels are 4-pixel (x-y) binned.
Fig. S15. Metabolic 2D ion images border the germinal center in labeled tonsil tissues. Mass-channel
numbers, along with the original 2D antibody labels and metabolites images obtained from reconstruction of
TOF-SIMS measurement data are shown for the outside of the germinal center tonsil dataset. The 2D images for
the selected mass-channels shown are 150-µm x 150-µm in area, reconstructed by using the Surfacelab version 6
software for the outside of the germinal center tonsil tissue dataset. The 2D image corresponding to the mass
channels are 4-pixel (x-y) binned.
Fig. S16. Network analysis of 3D metabolite and cell-specific features. A network-based visualization of
metabolites correlation. Edges represent correlation in red and anti-correlation in grey. Nodes in blue are the
metabolites and in red the markers. (A) Circular network with all the metabolites and markers. (B) Circular
network with all the metabolites and markers having correlation and anti-correlation edges. (C) Force network
with all the metabolites and markers having correlation and anti-correlation edges. (D) The circular network of
the markers.
Fig. S17. Network analysis of 3D metabolite and cell-specific features.
A network-based visualization of 3D metabolites and cell-specific correlations. Edges represent correlation in red
and anti-correlation in grey. Nodes in blue are the metabolites and in red the markers.
(A) The network inside the GC in tonsil tissues.
(B) The network at the border region of the GC tonsil tissues
(C) The network outside the GC tonsil tissues.
(D) The network of the unlabeled tonsil tissues.

Fig. S18. Spatial clustering by K-means and coloring of six clusters for all mass-channels.
A comparison of all mass-channel metabolic images spatial clustering for the inside, outside, and the border GC
is shown. Using K-means clustering to extract six clusters for all mass-channels, the average representation of
each cluster is shown with a unique color. Composite - Average Overlaid shows the 6 clusters representation
overlaid on each other with transparency to obtain a spatial map. Composite - Maximum Intensity extract the
color of the maximum intensity between the 6 clusters average representation to obtain a unique spatial map
between the six clusters
Fig. S19. Spatial clustering by isotope-labeled and other lipid fragments in all mass-channels inside GC.
Results of spatial clustering of isotope-labeled and other lipid fragments in all mass-channels from inside GC are
shown. Each image is associated with the corresponding cluster color corresponding to fig. S18.
Fig. S20. Spatial clustering and coloring of only lipid fragments inside GC.
Results of spatial clustering of only lipid fragments from inside GC are shown. The cluster overlaid image result
of the cluster is shown. Each image is associated with the corresponding cluster color.
Fig. S21. Spatial clustering and coloring of all-masses outside GC.
Results of spatial clustering of isotope-labeled and other lipid fragments in all mass-channels from outside GC
are shown. Each image is associated with the corresponding cluster color corresponding to fig. S18.
Fig. S22. Spatial clustering and coloring of only lipid fragments outside GC.
Results of spatial clustering of only lipid fragments from outside GC are shown. The cluster overlaid image result
Fig. S23. Spatial clustering and coloring of all mass-channels at the border of GC.
Results of spatial clustering of isotope-labeled and other lipid fragments in all mass-channels from border GC are
shown. Each image is associated with the corresponding cluster color corresponding to fig. S18.
Fig. S24. Spatial clustering and coloring of only lipid fragments at the border of GC.
Results of spatial clustering of only lipid fragments from border GC are shown. The cluster overlaid image result
Fig. S25. Optical image of isotope-labeled and unlabeled tissues before and after the measurements with
time-stamp information. The upper row shows the image at the start of the measurements and the bottom row
corresponds to the image at the end of the measurement for the datasets unlabeled and labeled of the GC of the
tonsil slice. The area of the field of view (FOV) captured in the entirety of each 2D image is 815 µm x 771 µm.
It can be observed that despite the 2D raster scanning of TOF-SIMS on the tonsil tissue, the images before
measurement minimally vary compared to the images captured after measurement.
Fig. S26. Spatial clustering and coloring of all mass-channels in a single LABELED GC with inside and
outside.
Results of spatial clustering of isotope-labeled and other mass channels in a single labeled GC with inside and
outside are shown. The cluster overlaid image result of the cluster is shown. Each image is associated with the
corresponding cluster color.
Fig. S27. Spatial clustering and coloring of only lipid fragments in a single LABELED GC with inside
and outside.
Results of spatial clustering of only lipid fragments in a single labeled GC with inside and outside are shown. The
cluster overlaid image result of the cluster is shown. Each image is associated with the corresponding cluster
color.
Fig. S28. Spatial clustering and coloring of all mass-channels in a single UNLABELED GC with inside
and outside.
Results of spatial clustering of mass channels corresponding to the isotope-labeled channel in Figure S26 and
other mass channels in a single unlabeled GC with inside and outside are shown. The cluster overlaid image result
Fig. S29. Spatial clustering and coloring of only lipid fragments in a single UNLABELED GC with inside
and outside. Results of spatial clustering of only lipid fragments in a single unlabeled GC with inside and outside
are shown. The cluster overlaid image result of the cluster is shown. Each image is associated with the
corresponding cluster color.
Fig. S30. Lipid codes inside and outside the germinal center using average signal values on a binary map
that is generated from germinal center boundaries.
(A) From the sum of the rest mass channel signal, the germinal center region is segmented for the labeled dataset.
(B) From the sum of the rest mass channel signal, the germinal center region is segmented for the unlabeled
dataset.
(C) Computed mean signal values inside and outside the germinal center for the labeled dataset are shown.
(D) Computed mean signal values inside and outside the germinal center for the unlabeled dataset are shown.
Fig. S31. Isotope labeling of the same GC increases the TOF-SIMS signal. (A) Comparison of the pixel
intensity level of TOF-SIMS signal for the total of identified mass-channel and the sum of the rest of the signal
for labeled and unlabeled GC. It is shown that Isotope labeling increases the TOF-SIMS signal (B) Comparison
of isotope-labeled channel sum of intensities. Isotope labeled channels have increased signal. (C) Comparison
of lipid fragment channel sum of intensities. Lipid fragments channel shows the variation in intensity
differences between labeled and unlabeled GC.
Fig. S32. Isotope labeling contributes to the amino-acid mass-channels from antibody-fragments.
(A) Mean intensity of TOF-SIMS signal for amino-acid mass-channels for the isotope-labeled and unlabeled
dataset
(B) TOF-SIMS signal image of amino-acid mass-channels for labeled GC dataset
(C) TOF-SIMS signal image of amino-acid mass-channels for unlabeled GC dataset

Fig. S33. Labeled images were subtracted from unlabeled images for the same GCs to show the distinct
images in isotope-specific, amino-acid, and lipid channels.
(A) TOF-SIMS signal differences between labeled images and unlabeled images from the same GCs for isotope-
labeled mass channels
(B)TOF-SIMS signal differences between labeled images and unlabeled images from the same GCs for amino-
acid mass channels
(C) TOF-SIMS signal differences between labeled images and unlabeled images from the same GCs for lipid
mass channels
Fig. S34. Isotope and antibody-positive enrichment factors by labeling and lipid positive and negative
variations that are calculated by labeled subtracted by unlabeled divided by labeled TOF-SIMS signals per
mass-channel.
This graph shows the relative intensity enrichment of TOF-SIMS signals per mass channel for the isotope-labeled
dataset compared to the unlabeled dataset. There is a positive signal enrichment related to isotope-labeled and
amino acid mass channels. For lipid mass channels, there is a variation between positive and negative enrichment
across mass channels. The enrichment is calculated by taking the mean of the subtraction labeled signal by the
unlabeled signal of the same mass channel divided by the unlabeled signal from TOF-SIMS.
Fig. S35. Spatial cross-correlation analyses reveal that amino-acids exhibit high-similarity due to shared
antibody fragments, possible common regulation of lipids, and negligible similarity of isotope-specific
channels due to the uniqueness of each label.
(A) Spatial cross-correlation analysis between isotope-labeled and unlabeled TOF-SIMS signal. First, the
difference of signal from each mass channel is obtained. The cross-correlation of signals from pairs of mass
channels is then calculated.
(B) Spatial cross-correlation result of all mass channels and a subset of the mass channel with the strongest
correlation. The analysis reveals that amino-acids exhibit high-similarity due to shared antibody fragments,
possible common regulation of lipids, and negligible similarity of isotope-specific channels due to the uniqueness
of each label.
Fig. S36. An inverse model that calculates the mean of similarity in amino-acid channels due to antibody
fragment contributions to predict a labeled channel from an unlabeled one. The antibody fragmentation
signals will be similar in multiple amino-acid channels and this similarity can be used to calculate potential
contributions on an unlabeled channel, yielding a predicted labeled amino-acid channel. Intentionally, the learning
of antibody channels was calculated from an image set that doesn’t contain the predicted channel.
(A) TOF-SIMS signal differences are calculated for amino-acid mass-channels except for the predicted mass
channel from the isotope-labeled and the unlabeled datasets.
(B) Mean intensity of the differences is calculated.
(C) Add to the unlabeled dataset predicted mass channel signal
(D) Prediction of the labeled mass channel signal

Fig. S37. Predicted mass-channel images provide a decent agreement to original mass-images of amino-
acid fragments due to antibody only contributions. The quantification was performed by a structural similarity
index (SSIM) that yielded a range of 0.2 to 0.43 values, indicating spatial similarity.
Table S1. Top 20 Metabolic 3D correlations for unlabeled tonsil tissues.
Compound 1 Compound 2 Correlation Coefficient
1 60 76 0.9691
2 16 17 0.9316
3 60 61 0.9193
4 60 16 0.9138
5 16 76 0.8988
6 50 185 0.8963
7 60 17 0.8948
8 61 76 0.8883
9 63 79 0.8878
10 269 76 0.8859
11 328 76 0.8819
12 269 60 0.8751
13 76 17 0.8653
14 201 185 0.8592
15 50 42 0.8581
16 60 328 0.8565
17 61 17 0.8562
18 50 201 0.8549
19 61 16 0.8516
20 76 179 0.8513
Table S2. Top 20 Metabolic 3D Anti-correlations for unlabeled tonsil tissues.
1 73 76 -0.431687
2 50 76 -0.426556
3 76 97 -0.416816
4 74 76 -0.415392
5 76 98 -0.415234
6 328 73 -0.390472
7 50 328 -0.383154
8 72 76 -0.379053
9 328 97 -0.373674
10 328 74 -0.371709
11 328 98 -0.369889
12 269 73 -0.356838
13 60 98 -0.35594
14 76 185 -0.355867
15 269 98 -0.348979
16 269 97 -0.347371
17 269 74 -0.346
18 50 269 -0.344572
19 50 60 -0.343333
20 60 74 -0.343136
Table S3. Top 20 Metabolic 3D correlations for inside germinal tonsil tissues.
1 75 84 0.849713
2 60 76 0.844244
3 75 91 0.836079
4 60 16 0.830711
5 16 17 0.821111
6 16 76 0.815416
7 75 42 0.810186
8 84 91 0.802364
9 35 42 0.801494
10 91 42 0.784948
11 75 86 0.780376
12 84 86 0.773837
13 50 75 0.771108
14 35 185 0.759734
15 75 35 0.755232
16 42 185 0.753863
17 84 42 0.748085
18 194 185 0.742028
19 194 35 0.738453
20 86 91 0.736214
Table S4. Top 20 Metabolic 3D Anti-correlations for inside germinal tonsil tissues.
1 50 16 -0.033556
2 50 179 -0.017323
3 50 76 -0.009228
4 16 122 -0.008594
5 198 1 -0.008122
6 289 1 -0.007437
7 359 1 -0.007232
8 1 131 -0.006887
9 1 80 -0.006778
10 240 1 -0.006701
11 1 185 -0.006666
12 273 1 -0.006409
13 344 1 -0.006049
14 267 1 -0.006035
15 193 1 -0.005864
16 529 1 -0.005672
17 229 1 -0.005614
18 1 138 -0.005592
19 412 544 -0.005538
20 498 1 -0.005471
Table S5. Top 20 Metabolic 3D correlations for outside germinal tonsil tissues.
1 75 42 0.687596
2 91 42 0.668263
3 75 91 0.663961
4 84 91 0.617178
5 84 42 0.613023
6 75 84 0.606509
7 63 79 0.588779
8 75 35 0.565462
9 35 42 0.548239
10 50 35 0.535107
11 79 128 0.531653
12 66 42 0.497375
13 91 100 0.497375
14 100 42 0.482626
15 66 75 0.482495
16 84 100 0.479357
17 66 91 0.474405
18 75 86 0.471822
19 86 91 0.470268
20 91 107 0.468618
Table S6. Top 20 Metabolic 3D Anti-correlations for outside germinal tonsil tissues.
1 73 75 -0.189442
2 73 42 -0.185055
3 73 91 -0.182625
4 1 35 -0.178095
5 1 75 -0.160561
6 73 84 -0.144273
7 50 1 -0.134964
8 73 100 -0.132616
9 73 107 -0.124657
10 1 42 -0.12036
11 66 73 -0.119494
12 72 79 -0.119076
13 253 75 -0.117963
14 72 91 -0.117628
15 205 75 -0.116935
16 73 35 -0.114955
17 73 86 -0.112285
18 229 75 -0.112058
19 253 91 -0.111061
20 72 100 -0.107312
Table S7. Top 20 Metabolic 3D correlations for border germinal tonsil tissues.
1 75 42 0.87459
2 50 185 0.849247
3 50 74 0.849168
4 75 91 0.837025
5 50 73 0.836012
6 91 42 0.834984
7 75 84 0.832497
8 50 72 0.819509
9 50 35 0.797866
10 50 97 0.791004
11 75 185 0.786067
12 84 42 0.784121
13 74 185 0.782264
14 73 74 0.781412
15 72 73 0.778788
16 50 60 0.778708
17 50 98 0.766728
18 66 75 0.764454
19 72 74 0.760524
20 73 185 0.758586
Table S8. Top 20 Metabolic 3D correlations for border germinal tonsil tissues.
1 1 158 -0.008261
2 1 155 -0.008154
3 1 145 -0.007327
4 208 1 -0.007078
5 267 1 -0.007077
6 1 149 -0.00694
7 552 1 -0.00672
8 246 1 -0.006241
9 1 107 -0.006166
10 1 116 -0.006094
11 276 1 -0.005813
12 1 30 -0.005682
13 1 134 -0.005669
14 1 83 -0.005393
15 1 85 -0.005357
16 1 104 -0.005335
17 1 78 -0.005123
18 1 150 -0.005014
19 479 1 -0.004998
20 1 162 -0.004571
Table S9. Chemical signatures assigned to selected compounds
Mass Number Compound General Category
1 42 CNO- Amino Acid
2 50 C3N- Rest
4 63 PO2- Rest
5 64 C4H2N- Rest
6 66 C5H7+ Lipid
7 72 C4H10N+ Amino Acid
8 73 C4H10N15+ Amino Acid
10 75 C4H12N15+ Rest
12 80 SO3- Rest
15 87 C5H12N15+ Rest
16 91 C7H7+ Lipid
17 93 C7H9+ Lipid
18 95 C7H11+ Lipid
19 97 C7H13+ Lipid
20 98 C5H8NO+ Rest
21 100 C5H10NO+ Lipid
23 107 Tyrosine Rest
24 111 C8H15+ Lipid
25 122 Cysteine Rest
26 127 I Rest
27 128 Serine Rest
28 131 Xe Rest
29 133 Asparagine Rest
30 138 Ba Rest
31 140 C2H7NO4P Rest
32 141 Smooth Muscle Actin Isotope-Labeled
33 143 Vimentin Isotope-Labeled
34 98 C5H8NO+ Lipid
35 146 C5H13NPO2+ Lipid
36 148 PanKertain Isotope-Labeled
37 150 PDL1 Isotope-Labeled
38 152 Gd Isotope-Labeled
39 155 Foxp3 Isotope-Labeled
40 156 CD4 Isotope-Labeled
41 158 E-Cadherin Isotope-Labeled
44 162 CD8A Isotope-Labeled
45 165 PD1 Isotope-Labeled
46 167 Granzyme B Isotope-Labeled
47 168 Ki 67 Isotope-Labeled
48 169 Collagen 1 Isotope-Labeled
50 171 Histone 3 Isotope-Labeled
51 173 CD45RO Rest

53 191 Intercalator(DNA) Isotope-Labelled
54 193 Intercalator(DNA) Isotope-Labeled
55 229 C14H27O2- Rest
57 385 C23H45O4- Lipid
Table S10. Lipidomic signatures assigned to mass numbers
Mass Number Compound General Category
1 30 C2D3+ Lipid
2 34 C2D5+ Lipid
3 55 C4H7+ Lipid
4 57 C4H9+ Lipid
5 58 C4D5+ Lipid
6 59 C3H8N15+ Lipid
7 60 C3H10N+ Lipid
8 66 C5H7+ Lipid
9 67 C5H7+ Lipid
10 68 C4H6N+ Lipid
11 69 C5H9 Lipid
12 71 C5H11+ Lipid
13 81 C6H9+ Lipid
14 83 C6H11+ Lipid
15 85 C6H13+ Lipid
16 88 C5H14N+ Lipid
17 91 C7H7+ Lipid
18 93 C7H9+ Lipid
19 95 C7H11+ Lipid
20 97 C7H13+ Lipid
21 98 C5H8N15O- Lipid
25 105 C5H14N15O+ Lipid
26 109 C8H13 Lipid
27 111 C8H15+ Lipid

37 205 C13H17O2 Lipid
38 206 C5H14NPO4Na+ Lipid
47 246 C8H18NPO4Na+ Lipid

49 253 C16H29O2 Lipid
53 283 C18H35O2 Lipid
54 289 C15H13O6 Lipid
55 301 C15H9O7 Lipid
56 339 C21H39O3 Lipid
57 369 C27H45 Lipid
58 385 C23H45O4- Lipid
59 431 C21H19O10 Lipid
60 521 C33H61O4 Lipid
Table S11. Lipid mass corresponding classification from LIPID MAP
Mass LIPID MAP

55
56
57
58 Oxygenated hydrocarbons [FA12]
59
Fatty alcohols [FA05]
60 Fatty Acids and Conjugates [FA01] - Straight chain fatty acids [FA0101]
66
67
68
69
70 Oxygenated hydrocarbons [FA12]
71
72
Oxygenated hydrocarbons [FA12]
Hydrocarbons [FA11]
74 Fatty Acids and Conjugates [FA01] - Unsaturated fatty acids [FA0103]
81
82
83
84 Hydrocarbons [FA11]
85
Hydrocarbons [FA11]
Fatty esters [FA07] - Lactones [FA0704]
Fatty aldehydes [FA06]
Fatty esters [FA07] - Wax monoesters [FA0701]
Fatty Acids and Conjugates [FA01] - Oxo fatty acids [FA0106] - Branched fatty acids [FA0102] -
88 Straight chain fatty acids [FA0101]
91
93
95
97
Hydrocarbons [FA11]
Hydrocarbons [FA11]
Fatty esters [FA07] - Lactones [FA0704] - Wax monoesters [FA0701]
Fatty Acids and Conjugates [FA01] - Branched fatty acids [FA0102]
Non-ribosomal peptide/polyketidehybrids [PK14]

Fatty Acids and Conjugates [FA01] - Oxo fatty acids [FA0106] - Hydroxy fatty acids [FA0105] -
Unsaturated fatty acids [FA0103] - Branched fatty acids [FA0102] - Straight chain fatty acids
102 [FA0101]
Fatty Acids and Conjugates [FA01] - Dicarboxylic acids [FA0117] - Amino fatty acids [FA0110] -
104 Hydroxy fatty acids [FA0105]
105
111
Fatty Acids and Conjugates [FA01] - Dicarboxylic acids [FA0117] - Hydroxy fatty acids [FA0105] -
146 Branched fatty acids [FA0102]
148 Fatty Acids and Conjugates [FA01] - Oxo fatty acids [FA0106] - Hydroxy fatty acids [FA0105]
Isoprenoids [PR01] - C10 isoprenoids (monoterpenes) [PR0102]
Fatty Acids and Conjugates [FA01] - Halogenated fatty acids [FA0109] - Unsaturated fatty acids
166 [FA0103]
167
Hydrocarbons [FA11]
182 Fatty Acids and Conjugates [FA01] - Oxo fatty acids [FA0106] - Unsaturated fatty acids [FA0103]
Fatty Acids and Conjugates [FA01] - Carbocyclic fatty acids [FA0114] - Oxo fatty acids [FA0106] -
184 Hydroxy fatty acids [FA0105] - Unsaturated fatty acids [FA0103] - Branched fatty acids [FA0102]
185 Fatty amides [FA08] - Fatty acyl-homoserine lactones [FA0803]
Isoprenoids [PR01] - C40 isoprenoids (tetraterpenes) [PR0107]
Hydrocarbons [FA11]
Fatty Acids and Conjugates [FA01] - Dicarboxylic acids [FA0117] - Halogenated fatty acids [FA0109]
190 - Epoxy fatty acids [FA0107] - Hydroxy fatty acids [FA0105] - Unsaturated fatty acids [FA0103]
Hydrocarbons [FA11]
Fatty esters [FA07] - Lactones [FA0704]
Fatty Acids and Conjugates [FA01] - Dicarboxylic acids [FA0117]
194 Other Fatty Acyls [FA00]
Fatty Acids and Conjugates [FA01] - Halogenated fatty acids [FA0109] - Epoxy fatty acids [FA0107] -
196 Oxo fatty acids [FA0106] - Unsaturated fatty acids [FA0103] - Unsaturated fatty acids [FA0103]
Fatty Acids and Conjugates [FA01] - Carbocyclic fatty acids [FA0114] - Halogenated fatty acids
198 [FA0109] - Unsaturated fatty acids [FA0103] - Branched fatty acids [FA0102]
Quinones and hydroquinones [PR02] - Ubiquinones [PR0201]
Isoprenoids [PR01] - C15 isoprenoids (sesquiterpenes) [PR0103]
Hydrocarbons [FA11]
Fatty Acids and Conjugates [FA01] - Carbocyclic fatty acids [FA0114] - Thia fatty acids [FA0113] -
206 Halogenated fatty acids [FA0109] - Epoxy fatty acids [FA0107] - Hydroxy fatty acids [FA0105]
Octadecanoids [FA02] - Jasmonic acids [FA0202]
Fatty Acids and Conjugates [FA01] - Dicarboxylic acids [FA0117] - Heterocyclic fatty acids [FA0115]
- Halogenated fatty acids [FA0109] - Unsaturated fatty acids [FA0103]
Fatty Acids and Conjugates [FA01] - Carbocyclic fatty acids [FA0114] - Oxo fatty acids [FA0106] -
212 Hydroxy fatty acids [FA0105] - Unsaturated fatty acids [FA0103] - Branched fatty acids [FA0102]
Fatty Acids and Conjugates [FA01] - Heterocyclic fatty acids [FA0115] - Oxo fatty acids [FA0106] -
Unsaturated fatty acids [FA0103] - Branched fatty acids [FA0102]
Fatty Acids and Conjugates [FA01] - Dicarboxylic acids [FA0117] - Heterocyclic fatty acids [FA0115]
- Carbocyclic fatty acids [FA0114] - Halogenated fatty acids [FA0109] - Unsaturated fatty acids
[FA0103] -
226 Branched fatty acids [FA0102]
Octadecanoids [FA02] - 12-oxophytodienoic acid metabolites [FA0201]
Fatty Acids and Conjugates [FA01] - Heterocyclic fatty acids [FA0115] - Oxo fatty acids [FA0106] -
238 Unsaturated fatty acids [FA0103]
Octadecanoids [FA02] - 12-oxophytodienoic acid metabolites [FA0201]
Fatty Acids and Conjugates [FA01] - Heterocyclic fatty acids [FA0115] - Carbocyclic fatty acids
[FA0114] - Oxo fatty acids [FA0106] - Unsaturated fatty acids [FA0103] - Branched fatty acids
240 [FA0102]
Isoprenoids [PR01] - C15 isoprenoids (sesquiterpenes) [PR0103] - C5 isoprenoids (hemiterpenes)
[PR0101]
Fatty acyl glycosides [FA13] - Ascarosides [FA1304]
Hydrocarbons [FA11]
Fatty Acids and Conjugates [FA01] - Fatty Acids and Conjugates [FA01] - Carbocyclic fatty acids
246 [FA0114] - Halogenated fatty acids [FA0109] - Unsaturated fatty acids [FA0103]
252 [FA0114] - Unsaturated fatty acids [FA0103]
[FA0114] - Hydroxy fatty acids [FA0105] - Unsaturated fatty acids [FA0103] - Branched fatty acids
254 [FA0102]
Flavonoids [PK12] - Isoflavonoids [PK1205] - Flavans, Flavanols, and Leucoanthocyanidins [PK1202]
Fatty Acids and Conjugates [FA01] - Heterocyclic fatty acids [FA0115] - Methoxy fatty acids
[FA0108] - Oxo fatty acids [FA0106] - Branched fatty acids [FA0102] - Straight chain fatty acids
256 [FA0101]
[FA0114] - Halogenated fatty acids [FA0109] - Methoxy fatty acids [FA0108] - Hydroxy fatty acids
[FA0105] - Hydroperoxy fatty acids [FA0104] - Unsaturated fatty acids [FA0103] - Branched fatty
282 acids [FA0102]
Data file S1. List of mass-numbers compiled from previous TOF-SIMS literature. (MS Excel spreadsheet)
Data file S2. List of mass-numbers identified by Surface Lab software. (MS Excel spreadsheet)

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advances.sciencemag.

Supplementary Materials for

Spatially resolved 3D metabolomic profiling in tissues

*Corresponding author. Email: ahmet.coskun@bme.gatech.edu

Published 27 January 2021, Sci. Adv. 7, eabd0957 (2021)

The PDF file includes:

Other Supplementary Material for this manuscript includes the following:

Data files S1 and S2

(A) The network inside the GC in tonsil tissues.

(B) The network at the border region of the GC tonsil tissues

(C) The network outside the GC tonsil tissues.

(D) The network of the unlabeled tonsil tissues.

(B) TOF-SIMS signal image of amino-acid mass-channels for labeled GC dataset

(C) TOF-SIMS signal image of amino-acid mass-channels for unlabeled GC dataset

(C) Add to the unlabeled dataset predicted mass channel signal

(D) Prediction of the labeled mass channel signal

Compound 1 Compound 2 Correlation Coefficient

14 201 185 0.8592

Table S2. Top 20 Metabolic 3D Anti-correlations for unlabeled tonsil tissues.

Compound 1 Compound 2 Correlation Coefficient

Compound 1 Compound 2 Correlation Coefficient

18 194 185 0.742028

Compound 1 Compound 2 Correlation Coefficient

19 412 544 -0.005538

Compound 1 Compound 2 Correlation Coefficient

Compound 1 Compound 2 Correlation Coefficient

Compound 1 Compound 2 Correlation Coefficient

Mass Number Compound General Category

1 42 CNO- Amino Acid

7 72 C4H10N+ Amino Acid

8 73 C4H10N15+ Amino Acid

9 74 C4H12N+ Amino Acid

13 84 C4H10N+ Amino Acid

14 86 C6H12N+ Amino Acid

21 100 C5H10NO+ Lipid

22 102 C5H12NO+ Lipid

23 107 Tyrosine Rest

24 111 C8H15+ Lipid

25 122 Cysteine Rest

27 128 Serine Rest

29 133 Asparagine Rest

31 140 C2H7NO4P Rest

32 141 Smooth Muscle Actin Isotope-Labeled

33 143 Vimentin Isotope-Labeled

36 148 PanKertain Isotope-Labeled

37 150 PDL1 Isotope-Labeled

39 155 Foxp3 Isotope-Labeled

40 156 CD4 Isotope-Labeled

41 158 E-Cadherin Isotope-Labeled

42 159 CD68 Isotope-Labeled

43 161 CD20 Isotope-Labeled

44 162 CD8A Isotope-Labeled

45 165 PD1 Isotope-Labeled

46 167 Granzyme B Isotope-Labeled

48 169 Collagen 1 Isotope-Labeled

49 170 CD3 Isotope-Labeled

50 171 Histone 3 Isotope-Labeled

51 173 CD45RO Rest

52 190 C7H13NPO3+ Lipid

54 193 Intercalator(DNA) Isotope-Labeled

55 229 C14H27O2- Rest

56 256 C8H19NPO6+ Lipid