Supplementary Figure 1 and 2: MALDI-IMS analysis in negative ion mode of 90-year-old human donor eye from fresh-frozen sample vs paraformaldehyde-fixed in peripheral sections from the same donor. Panels A-D display multiple lipid signals in both polarities in fixed and fresh-frozen tissue overlaid with an optical image of the tissues imaged. Varying signal intensities from the selected species can be seen for both fresh-frozen and fixed tissue.

Supplementary Figure 3: Positive ion mode MALDI-IMS of fresh-frozen vs fixed tissue displaying A PC(32:0), B PC(34:1) and C PC(34:2) from Figure 2D displaying the protonated, sodiated and potassiated ions which show the same localization but varying intensities.

Supplementary Figure 4: Positive ion mode MALDI-IMS of fresh-frozen vs fixed tissue displaying A TG(52:2) and B TG(52:3) displaying the protonated, sodiated and potassiated ions in fixed and fresh-frozen tissue overlaid with an optical image of the tissues imaged and MALDI IMS alone. These lipids show a high degree of localization in the choroid region with varying intensities.

Supplementary Figure 5: Global averaged mass spectra from negative and positive ion mode analysis of fixed and fresh tissues exported from imaging data. Panel 1A displays the negative ion mode mass spectrum from fixed tissue. Panel 1B displays the negative ion mode mass spectrum from fresh tissue. Panel 2A shows the positive ion mode mass spectrum from fixed tissue. Panel 2B shows the positive ion mode mass spectrum from fresh tissue.

Supplementary Figure 6: Replicate imaging mass spectrometry (IMS) data analysis of 84 year old human donor eye from fresh-frozen sample vs paraformaldehyde-fixed in peripheral sections from the same donor. Panels 1A (top, fixed) and B (bottom, fresh-frozen) display zoomed PASH stained tissue from an adjacent section indicating cell layers, NFL nerve fiber layer, GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, HFL Henle fiber layer, ONL outer nuclear layer, IS+OS inner segment + outer segment, RPE-BrM retinal pigment epithelium-Bruch’s membrane, Ch choroid. Panels 2A-D display multiple lipid signals in both polarities in fixed and fresh-frozen tissue. Panel A and B display negative ion mode analysis were three signals which were observed to have higher signal intensity in in fixed tissue (A) and fresh-frozen tissue (B). Panel C and D display negative ion mode analysis were three signals which were observed to have higher signal intensity in in fixed tissue (C) and fresh-frozen tissue (D). The poor morphology of the fresh-frozen tissue if reflected in the image quality in this example.

Supplementary Figure 7: Panel A and B show the differences in lipid composition from fixed (gray bars) and fresh-frozen (yellow bars) tissues analyzed using MALDI-IMS displaying multiple lipid classes observed in negative (Panel A) and positive (Panel B) ion mode analysis.

Supplementary Figure 8 A: Molecular ion spectra and fragmentation pattern in negative ionization mode of fixed tissue using LC-ESI-MS/MS. Calculated monoisotopic mass A1. 701.5121; PA (18:1_18:0); ppm error: 1.0, A2. 772.5281; PE(O-18:1_22:6); ppm error: 0.6 and A3. 1473.981; CL (18:2_20:3_18:2_18:2); ppm error: 1.3. B: Molecular ion spectra and fragmentation pattern in negative ionization mode of fresh-frozen tissue using LC-ESI-MS/MS. Calculated monoisotopic mass B1. 700.5281; (a) PE(O-16:0_18:2) and (b) PE(P-18:1_16:0) ; ppm error: 0.1, B2. 722.5125; (a) PE(O-16:0_20:5) and (b) PE(P-16:0_20:4); ppm error: 0.8, B3. 750.5443; (a) PE(O-16:0_22:5) and (b) PE(P-18:0_20:4); ppm error: 0.3. C: Molecular ion spectra and fragmentation pattern in positive ionization mode of fixed tissue using LC-ESI-MS/MS. Calculated monoisotopic mass C1. [M+H]+ 786.6013; PC(36:2) ; ppm error:2.6, C2. [M+H]+ 788.6169; PC(36:1); ppm error:0.6 , C3. [M+H]+ 814.6326; PC(38:2); ppm error: 5.6. D; Molecular ion spectra and fragmentation pattern in positive ionization mode of fixed tissue using LC-ESI-MS/MS. Calculated monoisotopic mass D1. [M+H]+ 734.5694; PC(32:0); ppm error: 1.9, D2. [M+H]+ 760.5851; PC(34:1); ppm error: 2.2, D3. [M+H]+ 786.6007; PC(36:2); ppm error: 1.9.

Supplementary Figure 9: LC-MS/MS data of fresh-frozen (blue boxes) vs fixed (red boxes) human donor retina (biological replicate 2). M/z versus retention time summary of (a) negative ion mode data and m/z versus retention time summary of (b) positive ion mode data where lipid classes.

Supplementary Figure 10: LC-MS/MS data of fresh-frozen (blue boxes) vs fixed (red boxes) human donor retina (biological replicate 3). M/z versus retention time summary of (a) negative ion mode data and m/z versus retention time summary of (b) positive ion mode data where lipid classes.

Supplementary Figure 11: Lipid composition identified (MS2 confirmations) in fresh-frozen and fixed human retina from LC-MS/MS using LipiDex.

Supplementary Table 1: LC-MS/MS signals for the IMS identified lipids as represented in figure 2. Three biological replicates were used in fresh-frozen and fixed retina in both ionization modes.

Supplementary Table 2: Comparison of intensities from fresh-frozen and fixed retina in representative lipids from each subclass investigated in the study. Average of 3 biological replicates were used for fresh-frozen and fixed retina in negative and positive ionization modes.

Supplementary Table 3: IMS identified lipids search results from Lipid Maps library in fresh-frozen and fixed retina tissue as identified in positive and negative ionization modes.