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MALDI IMS signals consistent with localization to photoreceptor and RPE compartments. (A) Schematic diagram of outer retina and Bruch's membrane, excerpted from Figure 2A. Blue, pink, yellow, and green bands indicate layers formed by highly compartmentalized and vertically aligned photoreceptors and RPE cells in panels B and C. See Figure 2 for explanation of cellular and subcellular content of each layer. Layers: OPL, outer plexiform layer; ONL, outer nuclear layer; ELM, external limiting membrane; RPE, retinal pigment epithelium; BrM, Bruch's membrane; R, Rod; C, cone photoreceptors. (B-F) Images and H&E stained tissue images overlaid in peripheral retina displaying signals from multiple lipid classes that localize to subcellular compartments of the photoreceptor cells. (B) Overlay showing four separate signals defined in panels C-F. (C) Localized to ONL. (D) Localized to photoreceptor inner and outer segments. (E) Localized to mitochondria-rich photoreceptor inner segments. (F) Localized to RPE apical processes.
Source publication
The human retina provides vision at light levels ranging from starlight to sunlight. Its supporting tissues regulate plasma-delivered lipophilic essentials for vision, including retinoids. The macula is an anatomic specialization for high-acuity and color vision that is also vulnerable to prevalent blinding diseases. The retina’s exquisite architec...
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... are the dominant photoreceptor in human retina (20:1 rod:cone ratio), being absent only in the coneonly foveal center. Signals unique to photoreceptor cells, a population necessarily dominated by rods in cross sections not including the fovea, are shown in Figure 3. Figure 3A shows part of the schematic from Figure 2A, focusing on photoreceptors and support cells. ...
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... are the dominant photoreceptor in human retina (20:1 rod:cone ratio), being absent only in the coneonly foveal center. Signals unique to photoreceptor cells, a population necessarily dominated by rods in cross sections not including the fovea, are shown in Figure 3. Figure 3A shows part of the schematic from Figure 2A, focusing on photoreceptors and support cells. The RPE sends delicate processes in the apical direction to contact photoreceptor OS, near the RPE cell body for rods and 10-15 μm above the cell body for cones (because cone OS are short). ...
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... RPE sits on Bruch's membrane, which serves as a flat vessel wall above the choriocapillaris endothelium. Figure 3A is color-coded to indicate photoreceptor and RPE compartments associated with IMS signals in Figure 3B (blue, red, yellow, green, for ONL, IS, OS, and RPE, respectively). is observed with high abundance in the ONL, which contains photoreceptor cell bodies and processes of Müller glia. The m/z 818.575 signal was assigned to photoreceptors due to the lack of similar signal in other retinal layers where Müller glia are also present. ...
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... RPE sits on Bruch's membrane, which serves as a flat vessel wall above the choriocapillaris endothelium. Figure 3A is color-coded to indicate photoreceptor and RPE compartments associated with IMS signals in Figure 3B (blue, red, yellow, green, for ONL, IS, OS, and RPE, respectively). is observed with high abundance in the ONL, which contains photoreceptor cell bodies and processes of Müller glia. The m/z 818.575 signal was assigned to photoreceptors due to the lack of similar signal in other retinal layers where Müller glia are also present. ...
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... signal observed at m/z 728.596 (green) is localized above and within the RPE, as discussed further below. Replicate data from an 81-year-old donor eye can be seen in Supplemental Figure 3. The image of m/z 728.596 ...
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... closer examination of Figure 2 this signal appears to extend above the RPE layer by up to 10 μm, depending on retinal position, with the greatest extensions observed at the fovea. Conversely, Figure 3 and Supplemental Figure 5 show signals possibly localized to only apical processes, i.e., not in the cell bodies, especially where apical processes are fortuitously standing upright. These discrepancies in m/z localizations are due to factors that impact alignment accuracy, e.g., OS attachment to RPE, angle of sectioning plane relative to the cell layer, and the different resolutions used (10 μm for Figures 2-5 vs 15 μm for Supplemental Figures 2 and 3). ...
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... A1, 2, and 4 also have strong signal in the HFL, NFL, and IPL extending across the whole macula. Figure 4A3 displays a similar overall pattern with high signal in the peripheral retina but lower signal in the central macula. In all four panels, no signal is observed in RPE directly below the signal-rich area of neurosensory retina, yet signals are intense in peripheral RPE. ...
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... signal observed at m/z 716.526 in Figure 4B1 was attributed to two phosphatidylethanolamine lipids PE(16:0_18:1) and PE(16:1_18:0) that are exact isobars, as confirmed in LC-MS/MS experiments. The MALDI IMS image generated from three phosphatidylinositol lipids, one tentatively identified as PI(32:1) ( Figure 4B2) and the other two identified as PI(16:0_18:2) and PI(18:0_18:2) ( Figure 4B3,4) all display the same distribution but with lower relative intensities than that observed for the PE lipid in Figure 4B1. The positively identified PI lipids contain an 18:2 fatty acid side chain. ...
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... are the dominant photoreceptor in human retina (20:1 rod:cone ratio), being absent only in the coneonly foveal center. Signals unique to photoreceptor cells, a population necessarily dominated by rods in cross sections not including the fovea, are shown in Figure 3. Figure 3A shows part of the schematic from Figure 2A, focusing on photoreceptors and support cells. ...
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... are the dominant photoreceptor in human retina (20:1 rod:cone ratio), being absent only in the coneonly foveal center. Signals unique to photoreceptor cells, a population necessarily dominated by rods in cross sections not including the fovea, are shown in Figure 3. Figure 3A shows part of the schematic from Figure 2A, focusing on photoreceptors and support cells. The RPE sends delicate processes in the apical direction to contact photoreceptor OS, near the RPE cell body for rods and 10-15 μm above the cell body for cones (because cone OS are short). ...
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... RPE sits on Bruch's membrane, which serves as a flat vessel wall above the choriocapillaris endothelium. Figure 3A is color-coded to indicate photoreceptor and RPE compartments associated with IMS signals in Figure 3B (blue, red, yellow, green, for ONL, IS, OS, and RPE, respectively). is observed with high abundance in the ONL, which contains photoreceptor cell bodies and processes of Müller glia. The m/z 818.575 signal was assigned to photoreceptors due to the lack of similar signal in other retinal layers where Müller glia are also present. ...
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... RPE sits on Bruch's membrane, which serves as a flat vessel wall above the choriocapillaris endothelium. Figure 3A is color-coded to indicate photoreceptor and RPE compartments associated with IMS signals in Figure 3B (blue, red, yellow, green, for ONL, IS, OS, and RPE, respectively). is observed with high abundance in the ONL, which contains photoreceptor cell bodies and processes of Müller glia. The m/z 818.575 signal was assigned to photoreceptors due to the lack of similar signal in other retinal layers where Müller glia are also present. ...
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... signal observed at m/z 728.596 (green) is localized above and within the RPE, as discussed further below. Replicate data from an 81-year-old donor eye can be seen in Supplemental Figure 3. The image of m/z 728.596 ...
Context 14
... closer examination of Figure 2 this signal appears to extend above the RPE layer by up to 10 μm, depending on retinal position, with the greatest extensions observed at the fovea. Conversely, Figure 3 and Supplemental Figure 5 show signals possibly localized to only apical processes, i.e., not in the cell bodies, especially where apical processes are fortuitously standing upright. These discrepancies in m/z localizations are due to factors that impact alignment accuracy, e.g., OS attachment to RPE, angle of sectioning plane relative to the cell layer, and the different resolutions used (10 μm for Figures 2-5 vs 15 μm for Supplemental Figures 2 and 3). ...
Context 15
... A1, 2, and 4 also have strong signal in the HFL, NFL, and IPL extending across the whole macula. Figure 4A3 displays a similar overall pattern with high signal in the peripheral retina but lower signal in the central macula. In all four panels, no signal is observed in RPE directly below the signal-rich area of neurosensory retina, yet signals are intense in peripheral RPE. ...
Context 16
... signal observed at m/z 716.526 in Figure 4B1 was attributed to two phosphatidylethanolamine lipids PE(16:0_18:1) and PE(16:1_18:0) that are exact isobars, as confirmed in LC-MS/MS experiments. The MALDI IMS image generated from three phosphatidylinositol lipids, one tentatively identified as PI(32:1) ( Figure 4B2) and the other two identified as PI(16:0_18:2) and PI(18:0_18:2) ( Figure 4B3,4) all display the same distribution but with lower relative intensities than that observed for the PE lipid in Figure 4B1. The positively identified PI lipids contain an 18:2 fatty acid side chain. ...
Citations
... There is some evidence that RPE under the macula lutea has a distinct lipid composition. 175 Combining the positive and negative effects creates a narrow center of foveal cone resilience amid a broader annular surround of parafoveal and perifoveal rod vulnerability (Fig. 9C). The result has a striking qualitative similarity to the annulus of photoreceptor degeneration and dysfunction in aging and early AMD. ...
A progression sequence for age-related macular degeneration onset may be determinable with consensus neuroanatomical nomenclature augmented by drusen biology and eye-tracked clinical imaging. This narrative review proposes to supplement the Early Treatment of Diabetic Retinopathy Study (sETDRS) grid with a ring to capture high rod densities. Published photoreceptor and retinal pigment epithelium (RPE) densities in flat mounted aged-normal donor eyes were recomputed for sETDRS rings including near-periphery rich in rods and cumulatively for circular fovea-centered regions. Literature was reviewed for tissue-level studies of aging outer retina, population-level epidemiology studies regionally assessing risk, vision studies regionally assessing rod-mediated dark adaptation (RMDA), and impact of atrophy on photopic visual acuity. The 3 mm-diameter xanthophyll-rich macula lutea is rod-dominant and loses rods in aging whereas cone and RPE numbers are relatively stable. Across layers, the largest aging effects are accumulation of lipids prominent in drusen, loss of choriocapillary coverage of Bruch's membrane, and loss of rods. Epidemiology shows maximal risk for drusen-related progression in the central subfield with only one third of this risk level in the inner ring. RMDA studies report greatest slowing at the perimeter of this high-risk area. Vision declines precipitously when the cone-rich central subfield is invaded by geographic atrophy. Lifelong sustenance of foveal cone vision within the macula lutea leads to vulnerability in late adulthood that especially impacts rods at its perimeter. Adherence to an sETDRS grid and outer retinal cell populations within it will help dissect mechanisms, prioritize research, and assist in selecting patients for emerging treatments.
... These technologies find broad applications in studying normal tissue development 1 , as well as in deciphering complex diseases like neurodegenerative disorders 2,3 and different types of cancer [4][5][6][7][8][9] . To investigate the various classes of molecules (RNA, proteins, metabolites, lipids) in a spatial context, different technologies are applied, including in situ hybridization, in situ sequencing and in situ RNA capturing followed by ex situ sequencing for spatial transcriptomics [10][11][12][13][14][15][16][17][18][19] (ST), histochemistry using fluorescently labelled antibodies [19][20][21] or genetically encoded fluorescent protein tags 22,23 for spatial proteomics, and mass spectrometry imaging (MSI) to study the distribution of peptides, metabolites, and lipids [24][25][26][27][28][29] . ...
Recent advances in spatial omics methods are revolutionising biomedical research by enabling detailed molecular analyses of cells and their interactions in their native state. As most technologies capture only a specific type of molecules, there is an unmet need to enable integration of multiple spatial-omics datasets. This, however, presents several challenges as these analyses typically operate on separate tissue sections at disparate spatial resolutions. Here, we established a spatial multi-omics integration pipeline enabling co-registration and granularity matching, and applied it to integrate spatial transcriptomics, mass spectrometry-based lipidomics, single nucleus RNA-seq and histomorphological information from human prostate cancer patient samples. This approach revealed unique correlations between lipids and gene expression profiles that are linked to distinct cell populations and histopathological disease states and uncovered molecularly different subregions not discernible by morphology alone. By its ability to correlate datasets that span across the biomolecular and spatial scale, the application of this novel spatial multi-omics integration pipeline provides unprecedented insight into the intricate interplay between different classes of molecules in a tissue context. In addition, it has unique hypothesis-generating potential, and holds promise for applications in molecular pathology, biomarker and target discovery and other tissue-based research fields.
... In this study, several typical marker compounds, mainly lipids, were found for each structure. An even more detailed study on the identification of lipids defining the retinal layers has been described by Anderson, Messinger, et al. (2020). Resolution of 5−10 µm allows to map drug distribution in the corneal (Mori et al., 2019) and retinal layers (Groseclose & Castellino, 2019). ...
Mass spectrometry (MS) has been proven as an excellent tool in ocular drug research allowing analyzes from small samples and low concentrations. This review begins with a short introduction to eye physiology and ocular pharmacokinetics and the relevance of advancing ophthalmic treatments. The second part of the review consists of an introduction to ocular proteomics, with special emphasis on targeted absolute quantitation of membrane transporters and metabolizing enzymes. The third part of the review deals with liquid chromatography–MS (LC‐MS) and MS imaging (MSI) methods used in the analysis of drugs and metabolites in ocular samples. The sensitivity and speed of LC‐MS make simultaneous quantitation of various drugs and metabolites possible in minute tissue samples, even though ocular sample preparation requires careful handling. The MSI methodology is on the verge of becoming as important as LC‐MS in ocular pharmacokinetic studies, since the spatial resolution has reached the level, where cell layers can be separated, and quantitation with isotope‐labeled standards has come more reliable. MS will remain in the foreseeable future as the main analytical method that will progress our understanding of ocular pharmacokinetics.
... With the application of matrix-assisted laser desorption/ ionization (MALDI) to imaging peptides and proteins in biological samples in the late 1990s (Caprioli et al., 1997), and the introduction of desorption electrospray ionization (DESI) in the early 2000s , MSI emerged as a powerful analytical approach. As a broadband label-free imaging approach, MSI allows spatial visualization of a variety of biological molecules, including proteins (Garza et al., 2018;Keener et al., 2021;Piehowski et al., 2020), peptides (Kakuda et al., 2017;Kaya et al., 2017), lipids (Anderson et al., 2020;Claes et al., 2021;Kaya et al., 2017;Unsihuay et al., 2020), polysaccharides (glycans) (Arnaud et al., 2020;Heijs, Holst-Bernal, et al., 2020;Heijs, Potthoff, et al., 2020;McDowell, Klamer, et al., 2021;McDowell, Lu, et al., 2021), amino acids (Esteve et al., 2016;J. He et al., 2019), and oligonucleotides (Nakashima & Setou, 2018;Yokoi et al., 2018) with softer ionization than preceding techniques such as secondary ion MS (SIMS). ...
Mass spectrometry (MS) has become a central technique in cancer research. The ability to analyze various types of biomolecules in complex biological matrices makes it well suited for understanding biochemical alterations associated with disease progression. Different biological samples, including serum, urine, saliva, and tissues have been successfully analyzed using mass spectrometry. In particular, spatial metabolomics using MS imaging (MSI) allows the direct visualization of metabolite distributions in tissues, thus enabling in‐depth understanding of cancer‐associated biochemical changes within specific structures. In recent years, MSI studies have been increasingly used to uncover metabolic reprogramming associated with cancer development, enabling the discovery of key biomarkers with potential for cancer diagnostics. In this review, we aim to cover the basic principles of MSI experiments for the nonspecialists, including fundamentals, the sample preparation process, the evolution of the mass spectrometry techniques used, and data analysis strategies. We also review MSI advances associated with cancer research in the last 5 years, including spatial lipidomics and glycomics, the adoption of three‐dimensional and multimodal imaging MSI approaches, and the implementation of artificial intelligence/machine learning in MSI‐based cancer studies. The adoption of MSI in clinical research and for single‐cell metabolomics is also discussed. Spatially resolved studies on other small molecule metabolites such as amino acids, polyamines, and nucleotides/nucleosides will not be discussed in the context.
... (16)(17)(18)(19) MALDI-IMS-driven lipidomics have also been central to both targeted and untargeted discoveries of lipid markers associated with pathophysiology, normal biochemical function, and metabolism across a wide range of model organisms and human tissues, with liquid chromatography tandem mass spectrometry (LC-MS/MS)-coupled approaches proving especially useful in lipid identification. (19)(20)(21)(22)(23) Despite this, MALDI-IMS approaches have seen limited application in bone primarily due to the challenging nature of this tissue; hydroxyapatite can hinder the identification of biomolecule distribution patterns. (24) However, proof-of-concept method development studies have demonstrated the feasibility of applying MALDI-IMS and similar techniques to bone and cartilage tissues, (25)(26)(27)(28) with recent advances enabling the biochemical fingerprinting of undecalcified tissues (29,30) and mapping the lipidomic changes in osteoarthritis (OA) synovial tissues. ...
... Moreover, there are distinct differences in the molecular composition of macular and peripheral RPE cells associated with differences in the abundance of overlying cones versus rods, respectively. [23][24][25][26] The complementarity between the macular cones and RPE cells 24 changes during development and aging, suggesting that macular RPE cells may have developed specifically for supporting cones. 25 If the macular RPE, because of its critical role in regulating the cone cell cycle and in supporting foveal cones, is implicit in forming and maintaining foveal shape, then a strong association between the distribution of cone and RPE cells and foveal shape in adolescents and adults would be expected. ...
... Moreover, there are distinct differences in the molecular composition of macular and peripheral RPE cells associated with differences in the abundance of overlying cones versus rods, respectively. [23][24][25][26] The complementarity between the macular cones and RPE cells 24 changes during development and aging, suggesting that macular RPE cells may have developed specifically for supporting cones. 25 If the macular RPE, because of its critical role in regulating the cone cell cycle and in supporting foveal cones, is implicit in forming and maintaining foveal shape, then a strong association between the distribution of cone and RPE cells and foveal shape in adolescents and adults would be expected. ...
... 25 The molecular composition of RPE cells differs between the macula and peripheral regions. [23][24][25][26] This lends support to the suggestion that the development and maturation of the RPE cell sheet could contribute to formation and maintenance of foveal shape and the considerable differences in peak cone density and topographies of foveal cones as observed in both ex vivo 38 and in vivo 52 studies. ...
Purpose:
To characterize the association between foveal shape and cone and retinal pigment epithelium (RPE) cell topographies in healthy humans.
Methods:
Multimodal adaptive scanning light ophthalmoscopy and optical coherence tomography (OCT) were used to acquire images of foveal cones, RPE cells, and retinal layers in eyes of 23 healthy participants with normal foveas. Distributions of cone and RPE cell densities were fitted with nonlinear mixed-effects models. A linear mixed-effects model was used to examine the relationship between cone and RPE inter-cell distances and foveal shape as obtained from the OCT scans of retinal thickness.
Results:
The best-fit model to the cone densities was a power function with a nasal-temporal asymmetry. There was a significant linear relationship among cone and RPE cell spacing, foveal shape, and foveal cell topography. The model predictions of the central 10° show that the contributions of both the cones and RPE cells are necessary to account for foveal shape.
Conclusions:
The results indicate that there is a strong relationship between cone and RPE cell spacing and the shape of the human adolescent and adult fovea. This finding adds to the existing evidence of the critical role that the RPE serves in fetal foveal development and through adolescence, possibly via the imposition of constraints on the number and distribution of foveal cones.
... 29 Mass spectrometry studies revealed differing lipid signals in RPE cells dependent on retinal locations. 30 Topography of AF and photoreceptors are congruent with increased AF in areas of high rod density. 17,31 Further, it has been shown that rod mediated vision loss precedes and exceeds those of cones. ...
Purpose:
Human retinal pigment epithelium (RPE) cells contain lipofuscin, melanolipofuscin, and melanosome organelles that impact clinical autofluorescence (AF) imaging. Here, we quantified the effect of age-related macular degeneration (AMD) on granule count and histologic AF of RPE cell bodies.
Methods:
Seven AMD-affected human RPE-Bruch's membrane flatmounts (early and intermediate = 3, late dry = 1, and neovascular = 3) were imaged at fovea, perifovea, and near periphery using structured illumination and confocal AF microscopy (excitation 488 nm) and compared to RPE-flatmounts with unremarkable macula (n = 7, >80 years). Subsequently, granules were marked with computer assistance, and classified by their AF properties. The AF/cell was calculated from confocal images. The total number of granules and AF/cell was analyzed implementing a mixed effect analysis of covariance (ANCOVA).
Results:
A total of 152 AMD-affected RPE cells were analyzed (fovea = 22, perifovea = 60, and near-periphery = 70). AMD-affected RPE cells showed increased variability in size and a significantly increased granule load independent of the retinal location (fovea: P = 0.02, perifovea: P = 0.04, and near periphery: P < 0.01). The lipofuscin fraction of total organelles decreased and the melanolipofuscin fraction increased in AMD, at all locations (especially the fovea). AF was significantly lower in AMD-affected cells (fovea: <0.01, perifovea: <0.01, and near periphery: 0.02).
Conclusions:
In AMD RPE, lipofuscin was proportionately lowest in the fovea, a location also known to be affected by accumulation of soft drusen and preservation of cone-mediated visual acuity. Enlarged RPE cell bodies displayed increased net granule count but diminished total AF. Future studies should also assess the impact on AF imaging of RPE apical processes containing melanosomes.
... Defining the cellular content of fibrotic scars is a key step in exploring new therapeutic targets to potentially reduce or prevent scar formation. New tissue-level techniques that reveal spatially resolved, multiplex molecular composition are becoming available (Anderson et al., 2020;Marx, 2021). Discovery of potential therapeutic targets with these methods can be accelerated by reference to detailed histology of intact donor eyes with nvAMD (Chen et al., 2020a;Curcio et al., 2015;Li et al., 2018a), such as we present herein. ...
... Our observations can also assist the interpretation of imaging outcomes in trials for RPE replacement therapies. Finally, detailed histology is helpful for interpreting spatially-resolved, multiplex molecular composition in complex AMD tissues, as is now technically feasible (Anderson et al., 2020;Marx, 2021). ...
Purpose
Melanotic cells with large spherical melanosomes, thought to originate from retinal pigment epithelium (RPE), are found in eyes with neovascular age-related macular degeneration (nvAMD). To generate hypotheses about RPE participation in fibrosis, we correlate histology to clinical imaging in an eye with prominent black pigment in fibrotic scar secondary to nvAMD.
Methods
Macular findings in a white woman with untreated inactive subretinal fibrosis due to nvAMD in her right eye were documented over 9 years with color fundus photography (CFP), fundus autofluorescence (FAF) imaging, and optical coherence tomography (OCT). After death (age 90 years), this index eye was prepared for light and electron microscopy to analyze 7 discrete zones of pigmentation in the fibrotic scar. In additional donor eyes with nvAMD, we determined the frequency of black pigment (n = 36 eyes) and immuno-labeled for retinoid, immunologic, and microglial markers (RPE65, CD68, Iba1, TMEM119; n = 3 eyes).
Results
During follow-up of the index eye, black pigment appeared and expanded within a hypoautofluorescent fibrotic scar. The blackest areas correlated to melanotic cells (containing large spherical melanosomes), some in multiple layers. Pale areas had sparse pigmented cells. Gray areas correlated to cells with RPE organelles entombed in the scar and multinucleate cells containing sparse large spherical melanosomes. In 94% of nvAMD donor eyes, hyperpigmentation was visible. Certain melanotic cells expressed some RPE65 and mostly CD68. Iba1 and TMEM119 immunoreactivity, found both in retina and scar, did not co-localize with melanotic cells.
Conclusion
Hyperpigmentation in CFP results from both organelle content and optical superimposition effects. Black fundus pigment in nvAMD is common and corresponds to cells containing numerous large spherical melanosomes and superimposition of cells containing sparse large melanosomes, respectively. Melanotic cells are molecularly distinct from RPE, consistent with a process of transdifferentiation. The subcellular source of spherical melanosomes remains to be determined. Detailed histology of nvAMD eyes will inform future studies using technologies for spatially resolved molecular discovery to generate new therapies for fibrosis. The potential of black pigment as a biomarker for fibrosis can be investigated in clinical multimodal imaging datasets.
... 1 In a typical workflow, MALDI-IMS detects exact masses of unknown molecules. [1][2][3][4] In contrast, LC-MS/MS facilitates structural elucidation by generating molecularly-specific fragmentation spectra. [1][2][3]5,6 Combining analyte localization with accurate mass measurements from MALDI-IMS 2,7,8 with structural characterization by LC-MS/MS 7,9 greatly enhances the breadth and depth of analysis of a tissue lipidome. ...
Imaging mass spectrometry (IMS) allows the location and abundance of lipids to be mapped across tissue sections of human retina. For reproducible and accurate information, sample preparation methods need to be optimized. Paraformaldehyde fixation of a delicate multilayer structure like human retina facilitates the preservation of tissue morphology by forming methylene bridge cross-links between formaldehyde and amine/ thiols in biomolecules; however, retina sections analyzed by IMS are typically fresh-frozen. To determine if clinically significant inferences could be reliably based on fixed tissue, we evaluated the effect of fixation on analyte detection, spatial localization, and introduction of artefactual signals. Hence, we assessed the molecular identity of lipids generated by matrix-assisted laser desorption ionization (MALDI-IMS) and liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) for fixed and fresh-frozen retina tissues in positive and negative ion modes. Based on MALDI-IMS analysis, more lipid signals were observed in fixed compared to fresh-frozen retina. More potassium adducts were observed in fresh-frozen tissues than fixed as the fixation process caused displacement of potassium adducts to protonated and sodiated species in ion positive ion mode. LC-MS/MS analysis revealed an overall decrease in lipid signals due to fixation that reduced glycerophospholipids and glycerolipids and conserved most sphingolipids and cholesteryl esters. The high quality and reproducible information from untargeted lipidomics analysis of fixed retina informs on all major lipid classes, similar to fresh-frozen retina, and serves as a steppingstone towards understanding of lipid alterations in retinal diseases.
... Future studies could focus on the contribution of the different photoreceptor systems (cones versus rods) to lipofuscin accumulation within the RPE cells and whether lipofuscin originating from cone outer segment tips differs from that of rod outer segment tips. Imaging mass spectrometric studies of photoreceptors and RPE cells might further help to clarify this (20). ...
Background:
Cells of the retinal pigment epithelium (RPE) accumulate different kinds of granules (lipofuscin, melanolipofuscin, melanosomes) within their cell bodies, with lipofuscin and melanolipofuscin being autofluorescent after blue light excitation. High amounts of lipofuscin granules within the RPE have been associated with the development of RPE cell death and age-related macular degeneration (AMD); however, this has not been confirmed in histology so far. Here, based on our previous dataset of RPE granule characteristics, we report the characteristics of RPE cells from human donor eyes that show either high or low numbers of intracellular granules or high or low autofluorescence (AF) intensities.
Methods:
RPE flatmounts of fifteen human donors were examined using high-resolution structured illumination microscopy (HR-SIM) and laser scanning microscopy (LSM). Autofluorescent granules were analyzed regarding AF phenotype and absolute number of granules. In addition, total AF intensity per cell and granule density (number of granules per cell area) were determined. For the final analysis, RPE cells with total granule number below 5th or above the 95th percentile, or a total AF intensity ± 1.5 standard deviations above or below the mean were included, and compared to the average RPE cell at the same location. Data are presented as mean ± standard deviation.
Results:
Within 420 RPE cells examined, 42 cells were further analyzed due to extremes regarding total granule numbers. In addition, 20 RPE cells had AF 1.5 standard deviations below, 28 RPE cells above the mean local AF intensity. Melanolipofuscin granules predominate in RPE cells with low granule content and low AF intensity. RPE cells with high granule content have nearly twice (1.8 times) as many granules as an average RPE cell.
Conclusions:
In normal eyes, outliers regarding autofluorescent granule load and AF intensity signals are rare among RPE cells, suggesting that granule deposition and subsequent AF follows intrinsic control mechanisms at a cellular level. The AF of a cell is related to the composition of intracellular granule types. Ongoing studies using AMD donor eyes will examine possible disease related changes in granule distribution and further put lipofuscińs role in aging and AMD further into perspective.
