[Show abstract][Hide abstract] ABSTRACT: Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) has the ability to provide an enormous amount of information on the abundances and spatial distributions of molecules within biological tissues. The rapid progress in the development of this technology significantly improves our ability to analyze smaller and smaller areas and features within tissues. The mammalian eye has evolved over millions of years to become an essential asset for survival, providing important sensory input of an organism's surroundings. The highly complex sensory retina of the eye is comprised of numerous cell types organized into specific layers with varying dimensions, the thinnest of which is the 10 μm retinal pigment epithelium (RPE). This single cell layer and the photoreceptor layer contain the complex biochemical machinery required to convert photons of light into electrical signals that are transported to the brain by axons of retinal ganglion cells. Diseases of the retina, including age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy, occur when the functions of these cells are interrupted by molecular processes that are not fully understood. In this report, we demonstrate the use of high spatial resolution MALDI IMS and FT-ICR tandem mass spectrometry in the Abca4 (-/-) knockout mouse model of Stargardt disease, a juvenile onset form of macular degeneration. The spatial distributions and identity of lipid and retinoid metabolites are shown to be unique to specific retinal cell layers.
Journal of the American Society for Mass Spectrometry 05/2014; · 3.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite to understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system.
[Show abstract][Hide abstract] ABSTRACT: Lipofuscin, an aging marker in the retinal pigment epithelium (RPE) associated with the development of age-related macular degeneration, is primarily characterized by its fluorescence. The most abundant component of RPE lipofuscin is N-retinylidene-N-retinylethanolamine (A2E) but its exact composition is not known due to the complexity of the RPE extract. In this study, we utilized MALDI imaging to find potential molecules responsible for lipofuscin fluorescence in RPE tissue from Abca4(-/-) , Sv129, and C57Bl6/J mice ages 2 and 6 month. To assert relationships, the individual images in the MALDI imaging datasets were correlated with lipofuscin fluorescence recorded from the same tissues following proper registration. Spatial correlation information, which is usually is lost in bioanalytics, pinpointed a relatively small number of potential lipofuscin components. The comparison of four samples in each condition further limited the possibility of false positives and provided various new, age- and strain-specific targets. Validating the usefulness of the fluorescence-enhanced imaging strategy, many known adducts of A2E were identified in the short list of lipofuscin components. These results provided evidence that mass spectrometric imaging can be utilized as a tool to begin to identify the molecular substructure of clinically-relevant diagnostic information. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Ultraviolet radiation (UVR) exposure is a major risk factor for age-related cataract, a protein aggregation disease of the human lens often involving the major proteins of the lens, the crystallins. γD-Crystallin (HγD-Crys) is abundant in the nucleus of the human lens and its folding and aggregation have been extensively studied. Previous work showed that HγD-Crys photo-aggregates in vitro upon exposure to UVA/UVB light and that its conserved tryptophans are not required for aggregation. Surprisingly, the tryptophan residues play a photo-protective role due to a distinctive energy transfer mechanism. HγD-Crys also contains 14 tyrosine residues, twelve of which are organized as six pairs. We investigated the role of the tyrosines of HγD-Crys by replacing pairs with alanines and monitoring photo-aggregation using light scattering and SDS-PAGE. Mutating both tyrosines in the Y16/Y28 pair to alanine slowed the formation of light scattering aggregates. Further mutant studies implicated Y16 as important for photo-aggregation. Mass spectrometry revealed that C18, in contact with Y16, is heavily oxidized during UVR exposure. Analysis of multiple mutant proteins by mass spectrometry suggested that Y16 and C18 likely participate in the same photochemical process. The UVR-induced aggregation was suppressed upon incubation with the lens chaperone αB-crystallin. The data suggest an initial photo-aggregation pathway for HγD-Crys in which excited state Y16 interacts with C18, initiating radical polymerization.
[Show abstract][Hide abstract] ABSTRACT: The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart's pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican's expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan ((tm1Zim)), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan ((tm1Zim)) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.
PLoS ONE 01/2014; 9(2):e89133. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background & Aims
The gastric cancer-causing pathogen Helicobacter pylori upregulates spermine oxidase (SMOX) in gastric epithelial cells, causing oxidative stress-induced apoptosis and DNA damage. A subpopulation of SMOXhighcells are resistant to apoptosis, despite their high levels of DNA damage. Because epidermal growth factor receptor (EGFR) activation can regulate apoptosis, we determined its role in SMOX-mediated effects.
SMOX, apoptosis, and DNA damage were measured in gastric epithelial cells from H pylori-infected Egfrwa5mice (which have attenuated EGFR activity), Egfr wild-typemice, or in infected cells incubated with EGFR inhibitors or deficient in EGFR. Phosphoproteomic analysis was performed. Two independent tissue microarrays containing each stage of disease, from gastritis to carcinoma, and gastric biopsies from Colombian and Honduran cohorts were analyzed by immunohistochemistry.
SMOX expression and DNA damage were decreased, and apoptosis increased in H pylori-infected Egfrwa5mice. H pylori-infected cells with deletion or inhibition of EGFR had reduced levels of SMOX, DNA damage, and DNA damagehigh apoptosislowcells. Phosphoproteomic analysis revealed increased EGFR and ERBB2 signaling. Immunoblot analysis demonstrated the presence of a phosphorylated (p)EGFR–ERBB2 heterodimer and pERBB2; knockdown of ErbB2 facilitated apoptosis of DNA damagehigh apoptosislowcells. SMOX was increased in all stages of gastric disease, peaking in tissues with intestinal metaplasia, whereas pEGFR, pEGFR–ERBB2, and pERBB2 were increased predominantly in tissues demonstrating gastritis or atrophic gastritis. Principal component analysis separated gastritis tissues from patients with cancer vs those without cancer. pEGFR, pEGFR–ERBB2, pERBB2, and SMOX were increased in gastric samples from patients whose disease progressed to intestinal metaplasia or dysplasia, compared with patients whose disease did not progress.
In an analysis of gastric tissues from mice and patients, we identified a molecular signature (based on levels of pEGFR, pERBB2, and SMOX) for the initiation of gastric carcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: Background: All thirteen known mammalian aquaporins have been detected in the eye. Moreover, aquaporins have been identified as playing essential roles in ocular functions ranging from maintenance of lens and corneal transparency to production of aqueous humor to maintenance of cellular homeostasis and regulation of signal transduction in the retina. Scope of Review: This review summarizes the expression and known functions of ocular aquaporins and discusses their known and potential roles in ocular diseases. Major Conclusions: Aquaporins play essential roles in all ocular tissues. Remarkably, not all aquaporin functions are as a water permeable channel and the functions of many aquaporins in ocular tissues remain unknown. Given their vital roles in maintaining ocular function and their roles in disease, aquaporins represent potential targets for future therapeutic development. General Significance: Since aquaporins play key roles in ocular physiology, an understanding of these functions is important to improving ocular health and treating diseases of the eye. It is likely that future therapies for ocular diseases will rely on modulation of aquaporin expression and/or function. This article is part of a Special Issue entitled Aquaporins.
Biochimica et Biophysica Acta 10/2013; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Non-enzymatic posttranslational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC-tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty four of thirty six sites identified on fifteen proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through β-elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein-protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Non-disulfide crosslinks were identified for the first time in lens tissue between βB2- & βB2-, βA4- & βA3-, γS -& βB1- and βA4- & βA4-crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long-lived proteins and that DHA and DHB-mediated protein crosslinking may the source of the long sought after non-disulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long-lived proteins of other tissues leading to protein aggregation diseases. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: The accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD) in humans. The exact composition of lipofuscin is not known but its best characterized component is N-retinylidene-N-retinylethanolamine (A2E), a byproduct of the retinoid visual cycle. Utilizing our recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS)-based technique to determine the spatial distribution of A2E, this study compares the relationships of lipofuscin fluorescence and A2E in the murine and human RPE on representative normal tissue. To identify molecules with similar spatial patterns, the images of A2E and lipofuscin were correlated with all the individual images in the MALDI-IMS dataset. In the murine RPE, there was a remarkable correlation between A2E and lipofuscin. In the human RPE, however, minimal correlation was detected. These results were reflected in the marked distinctions between the molecules that spatially correlated with the images of lipofuscin and A2E in the human RPE. While the distribution of murine lipofuscin showed highest similarities with some of the known A2E-adducts, the composition of human lipofuscin was significantly different. These results indicate that A2E metabolism may be altered in the human compared to the murine RPE.
Archives of Biochemistry and Biophysics 08/2013; · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The accumulation of lipofuscin in the retinal pigment epithelium (RPE) is a hallmark of aging in the eye. The best characterized component of lipofuscin is A2E, a bis-retinoid by-product of the normal retinoid visual cycle, which exhibits a broad spectrum of cytotoxic effects in vitro. The purpose of this study was to correlate the distribution of lipofuscin and A2E across the human RPE.
Lipofuscin fluorescence was imaged in flat-mounted RPE from human donors of various ages. The spatial distributions of A2E and its oxides were determined using matrix-assisted laser desorption-ionization imaging mass spectrometry (MALDI-IMS) on both flat-mounted RPE tissue sections and on retinal cross sections.
Our data support the clinical observations of strong RPE fluorescence, increasing with age, in the central area of the RPE. However, there was no correlation between the distribution of A2E and lipofuscin, as the levels of A2E were highest in the far periphery and decreased towards the central region. High-resolution MALDI-IMS of retinal cross sections confirmed the A2E localization data obtained in RPE flat-mounts. Singly- and doubly-oxidized A2E had distributions similar to A2E, but represented <10% of the A2E levels.
This report is the first description of the spatial distribution of A2E in the human RPE by imaging mass spectrometry. These data demonstrate that the accumulation of A2E is not responsible for the increase in lipofuscin fluorescence observed in the central RPE with aging.
[Show abstract][Hide abstract] ABSTRACT: MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for localizing both small molecules and intact proteins in a wide variety of tissue samples in both normal and diseased states. Identification of imaged signals in MALDI-IMS remains a bottleneck in the analysis and limits the interpretation of underlying biology of tissue specimens. In this work, spatially-directed tissue microextraction of intact proteins followed by LC-MS/MS with electron transfer dissociation (ETD) was used to identify proteins from specific locations in three tissue types; ocular lens, brain, and kidney. Detection limits were such that a 1 microliter extraction volume was sufficient to deliver proteins to the LC-MS/MS instrumentation with sufficient sensitivity to detect 50-100 proteins in a single experiment. Additionally, multiple modified proteins were identified; including truncated lens proteins that would be difficult to assign to an imaged mass using a bottom-up approach. Protein separation and identification are expected to improve with advances in intact protein fractionation/chromatography and advances in interpretation algorithms leading to increased depth of proteome coverage from distinct tissue locations.
[Show abstract][Hide abstract] ABSTRACT: The optic nerve is the conduit for transmitting visual signals along retinal ganglion cell axons to multiple sub-cortical nuclei involved in various vision-related tasks . These axons are unmyelinated in the retina and remain so in exiting the retina through the optic disc until passing through the optic nerve head . Similar to other nerves within the central nervous system, the optic nerve has associated meninges, including the arachnoidal and pial membranes and the dura, which blends into the sclera. Beyond the nerve head, the fibers become myelinated by oligodendrocytes  similar to the white matter in brain and spinal cord tissue . Lipids are a major component of myelin and meninges and support the functional activity of neuronal tissue; therefore, the lipid composition and spatial distribution of lipids in tissues are key to normal nerve function . Optic nerve lipid composition studies indicate that major lipid components include neutral lipid species such as cholesterol, glycosphingolipids such as cerebrosides, and several glycerophospholipids [5,6]. Changes in optic nerve structure and function are key contributors to glaucoma, the most common optic neuropathy, and include early insult to ganglion cell axons . Many pathogenic features of glaucoma involve spatially specific changes in nerve function and morphology [8,9]. Thus, understanding the spatial pattern of molecular changes is essential for probing molecular mechanisms of disease and identifying new therapeutic targets. Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is a novel imaging modality capable of providing molecular ion maps across tissue surfaces including maps of lipids, proteins, and metab-olites [10-12]. In a MALDI-IMS experiment, information about molecular distribution and relative intensity is acquired. In recent years, several papers on imaging mass spectrometry of ocular tissue, including lens, lens capsule, lens lipids, flat mounted retina, and retinal cross sections, have been published [13-19]. Since MALDI-IMS was developed in 1997 by the Caprioli group , the technology has progressed rapidly to allow for greater sensitivity and improved spatial resolution. The spatial resolution of MALDI-IMS is currently
[Show abstract][Hide abstract] ABSTRACT: Membrane proteins are abundant, critically important biomolecules that carry out essential functions in all cells and are the targets of a significant number of therapeutic drugs. However, their analysis by mass spectrometry to determine protein expression, protein modification, protein-protein interactions, and protein structure, has lagged behind similar studies of soluble proteins. Here we review the limitations to analysis of integral membrane and membrane-associated proteins and highlight advances in sample preparation and mass spectrometry methods that have led to the successful analysis of this protein class. Advances in the analysis of membrane protein posttranslational modification, protein-protein interaction, protein structure, and tissue distributions by imaging mass spectrometry are discussed. Furthermore, we focus our discussion on the application of mass spectrometry for the analysis of aquaporins as a prototypical integral membrane protein and how advances in analytical methods have revealed new biological insights into the structure and function of this family of proteins.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: The human lens fiber cell insoluble membrane fraction contains important membrane proteins, cytoskeletal proteins and cytosolic proteins that are strongly associated with the membrane. The purpose of this study was to characterize the lens fiber cell membrane proteome and phosphoproteome from human lenses. METHODS: HPLC-mass spectrometry based Multidimensional Protein Identification Technology (MudPIT), without or with phosphopeptide enrichment, was applied to study the proteome and phosphoproteome of lens fiber cell membranes, respectively. RESULTS: In total, 951 proteins were identified including 379 integral membrane and membrane-associated proteins. Enriched gene categories and pathways based on the proteomic analysis include carbohydrate metabolism (glycolysis/gluconeogenesis, pentose phosphate pathway, pyruvate metabolism), proteasome, cell-cell signaling and communication (GTP binding, gap junction, focal adhesion), glutathione metabolism, and actin regulation. The combination of TiO2 phosphopeptide enrichment and MudPIT analysis revealed 855 phosphorylation sites on 271 proteins including 455 phosphorylation sites that have not been previously identified. PKA, PKC, CKII, p38MAPK and RSK are predicted as the major kinases for phosphorylation on the sites identified in the human lens membrane fraction. CONCLUSIONS: The results presented herein significantly expand the characterized proteome and phosphoproteome of the human lens fiber cell and provide a valuable reference for future research in studies of lens development and disease.
[Show abstract][Hide abstract] ABSTRACT: Background: the required role of versican (Vcan gene) in early heart development has been demonstrated by analysis of the mouse mutant called heart defect (hdf). Embryos homozygous for the insertional mutation lack detectable versican protein and die at an early stage with severe heart defects that include the lack of prevalvular cushion tissue, dilated myocardium and an underdeveloped outflow tract. The early embryonic lethality of the hdf mice has complicated in vivo investigations into the functional role of versican at later more clinically relevant stages of heart development when versican is abundantly expressed. In this study we present proteomic data from a mouse model of heart defects that arise from the selective deletion of two (V2/V0) of the four alternative splice forms of versican.
Results: we find that mice homozygous for the deletion can survive into adulthood, however, the loss of V2/V0 splice form of versican results in embryonic heart septal defects (VSD) and dilated right ventricle. We correlate these phenotypic findings with large-scale differential protein expression profiling using iTRAQ-2DLC-MS/MS, to identify alterations in cardiac protein expression, such as Cypher/ZASP, annexins, vimentin associated with heart dysmorphogenesis.
Conclusions: the loss of versican V2/V0 splice forms result in significant alterations in cytoskeletal and contraction proteins in the heart that likely correlate with septal defects and dilated right ventricle.
[Show abstract][Hide abstract] ABSTRACT: Until recently, the lens was thought to express only two aquaporin (AQP) water channels, AQP1 and AQP0. In this study we confirm lenticular AQP5 protein expression by Western blotting and mass spectrometry in lenses from a variety of species. In addition, confocal microscopy was used to map cellular distributions of AQP5 in mouse, rat and human lenses. Tandem mass spectrometry of a human lens membrane preparation revealed extensive sequence coverage (56.2%) of AQP5. Western blotting performed on total fiber cell membranes from mouse, rat, bovine and human lenses confirmed AQP5 protein expression is conserved amongst species. Western blotting of dissected lens fractions suggests that AQP5 is processed in the lens core by C-terminal truncation. Immunohistochemistry showed that AQP5 signal was most abundant in the lens outer cortex and decreased in intensity in the lens core. Furthermore, AQP5 undergoes differentiation-dependent changes in subcellular location from an intracellular localization in differentiating fiber cells to the plasma membrane of mature fiber cells upon the loss of fiber cell nuclei. Our results show that AQP5 is a significant component of lens fiber cell membranes, representing the second most abundant water channel in these cells. Together, the changes to AQP5 distribution and structure are likely to modulate the functional role of AQP5 in different regions of the lens.
Experimental Eye Research 01/2013; · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations reveal a crucial role for LTCCs in regulation of expression, activity and stability of aquaporin-0, connexins, cytoskeletal proteins, and the mechanical properties of lens, all of which have a vital role in maintaining lens function and cytoarchitecture.
PLoS ONE 01/2013; 8(5):e64676. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To develop a method for generating high spatial resolution (10 µm) matrix-assisted laser desorption ionization (MALDI) images of lipids in rodent optic nerve tissue.
Ice-embedded optic nerve tissue from rats and mice were cryosectioned across the coronal and sagittal axes of the nerve fiber. Sections were thaw mounted on gold-coated MALDI plates and were washed with ammonium acetate to remove biologic salts before being coated in 2,5-dihydroxybenzoic acid by sublimation. MALDI images were generated in positive and negative ion modes at 10 µm spatial resolution. Lipid identification was performed with a high mass resolution Fourier transform ion cyclotron resonance mass spectrometer.
Several lipid species were observed with high signal intensity in MALDI images of optic nerve tissue. Several lipids were localized to specific structures including in the meninges surrounding the optic nerve and in the central neuronal tissue. Specifically, phosphatidylcholine species were observed throughout the nerve tissue in positive ion mode while sulfatide species were observed in high abundance in the meninges surrounding the optic nerve in negative ion mode. Accurate mass measurements and fragmentation using sustained off-resonance irradiation with a high mass resolution Fourier transform ion cyclotron resonance mass spectrometer instrument allowed for identification of lipid species present in the small structure of the optic nerve directly from tissue sections.
An optimized sample preparation method provides excellent sensitivity for lipid species present within optic nerve tissue. This allowed the laser spot size and fluence to be reduced to obtain a high spatial resolution of 10 µm. This new imaging modality can now be applied to determine spatial and molecular changes in optic nerve tissue with disease.