The surface oxidation potential of melanosomes measured by free electron laser-photoelectron emission microscopy

A technique for measuring the photoionization spectrum and the photoelectron emission threshold of a microscopic structured material is presented. The theoretical underpinning of the experiment and the accuracy of the measurements are discussed. The technique is applied to titanium silicide nanostructures and melanosomes isolated from human hair, human and bovine retinal pigment epithelium cells, and the ink sac of Sepia officinalis. A common photothreshold of 4.5 +/- 0.2 eV is found for this set of melanosomes and is attributed to the photoionization of the eumelanin pigment. The relationship between the photoionization threshold and the electrochemical potential referenced to the normal hydrogen electrode is used to quantify the surface oxidation potential of the melanosome. The developed technique is used to examine the effect of iron chelation on the surface oxidation potential of Sepia melanosomes. The surface oxidation potential is insensitive to bound Fe(III) up to saturation, suggesting that the metal is bound to the interior of the granule. This result is discussed in relation to the age-dependent accumulation of iron in human melanosomes in both the eye and brain.     

Effects of photodegradation on the physical and antioxidant properties of melanosomes isolated from retinal pigment epithelium

Melanosomes of the retinal pigment epithelium (RPE) are relatively long-lived organelles that are theoretically susceptible to changes induced by exposure to visible light. Here melanosomes were isolated from porcine RPE cells and subjected to high intensity visible light to determine the effects of illumination on melanosome structure and on the content and antioxidant properties of melanin. As compared to untreated melanosomes, illuminated granules showed morphologic changes consistent with photodegradation, which included variable reductions in electron density demonstrated by transmission electron microscopy (TEM), and particle fragmentation and surface disruption revealed by scanning electron microscopy (SEM) and atomic force microscopy. Illuminated melanosomes had lower melanin content, indicated by measures of absorbance and electron spin resonance (ESR) signal intensity, and reduced ability to bind iron, shown by chemical and ESR analyses. Compared to untreated melanosomes, ESR-spin trapping analyses further indicated that illuminated melanosomes show increased photogeneration of superoxide anion and reduced ability to inhibit the iron ion-catalyzed free radical decomposition of hydrogen peroxide. It appears therefore that visible light irradiation can disrupt the structure of RPE melanosomes and reduce the amount and antioxidant properties of melanin. Some of these changes occur in human RPE melanosomes with aging and the results obtained here suggest that visible light irradiation is at least partly responsible. The consequence of light-induced changes in RPE melanosomes may be a diminished capacity of melanin to help protect aged cells from oxidative damage, perhaps increasing the risk of diseases with an oxidative stress component such as age-related macular degeneration.     

Insights into melanosomes and melanin from some interesting spatial and temporal properties

Melanosomes are organelles found in a wide variety of tissues throughout the animal kingdom and exhibit a range of different shapes: spheres of up to approximately 1 mum diameters and ellipsoids with lengths of up to approximately 2 mum and varying aspect ratios. The functions of melanosomes include photoprotection, mitigation of the effects of reactive oxygen species, and metal chelation. The melanosome contains a variety of biological molecules, e.g., proteins and lipids, but the dominant constituent is the pigment melanin, and the functions ascribed to melanosomes are uniquely enabled by the chemical properties of the melanins they contain. In the past decade, there has been significant progress in understanding melanins and their impact on human health. While the molecular details of melanin production and how the pigment is organized within the melanosome determine its properties and biological functions, the physical and chemical properties of the surface of the melanosome are central to their range of ascribed functions. Surprisingly, few studies designed to probe this biological surface have been reported. In this article, we discuss recent work using surface-sensitive analytic, spectroscopic, and imaging techniques to examine the structural and chemical properties of many types of natural pigments: sepia melanin granules, human and bovine ocular melanosomes, human hair melanosomes, and neuromelanin. N 2 adsorption/desorption measurements and atomic force microscopy provide novel insights into surface morphology. The chemical properties of the melanins present on the surface are revealed by X-ray photoelectron spectroscopy and photoemission electron microscopy. These technologies are also applied to elucidate changes in surface properties that occur with aging. Specifically, studies of the surface properties of human retinal pigment epithelium melanosomes as a function of age are stimulating the development of models for their age-dependent behaviors. The article concludes with a brief discussion of important unanswered research questions in this field.     

Surface elastic properties of human retinal pigment epithelium melanosomes

Atomic force microscope (AFM) imaging and nanoindentation measurements in water were used to probe the mechanical properties of retinal pigment epithelium melanosomes isolated from 14-year-old and 76-year-old donors. Topographic imaging reveals surface roughness similar to previous measurements on dry melanosomes. Force-indentation measurements show different types of responses that were catalogued into four different categories. In these measurements no permanent surface damage of melanosomes was observed as revealed by imaging before and after indentation measurements. The indentation measurements that exhibited nearly elastic responses were used to determine the Young's modulus of melanosomes. The average Young's modulus values are similar for 14-year-old and 76-year-old melanosomes with a somewhat narrower distribution for the 14-year-old sample. These elastic modulus values are considerably higher than the modulus of organelles with cytoplasm (<1 MPa) and approaching values of the modulus of protein crystals (approximately 100 MPa) indicating rather high packing density of biologic material in melanosomes. The width of the Young's modulus distributions is considerable spanning from few megapascals to few tens of megapascals indicating large heterogeneity in the structure. A fraction of the force curves cannot be described by the homogeneous elastic sample model; these force curves are consistent with approximately 10 nm structural heterogeneity in melanosomes. The approach-withdraw hysteresis indicates a significant viscoelasticity, particularly in the samples from the 14-year-old sample. Adhesion of the AFM probe was detected on approximately 3% and approximately 20% of the surface of 14-year-old and 76-year-old samples, respectively. In light of previous studies on these same melanosomes using photoelectron emission microscopy, this adhesion is attributed to the presence of lipofuscin on the surface of the melanosomes. This suggestion indicates that part of the difference in photochemical properties between the old and young melanosomes originates from surface lipofuscin.     

AGE-RELATED CHANGES IN THE FLUORESCENCE OF MELANIN and LIPOFUSCIN GRANULES OF THE RETINAL PIGMENT EPITHELIUM: A TIME-RESOLVED FLUORESCENCE SPECTROSCOPY STUDY

The photophysical properties of purified populations of melanin and lipofuscin granules from human retinal pigment epithelium, and their changes with donor age, have been investigated using high-sensitivity time-resolved fluorescence spectroscopy techniques with picosecond gating capabilities. The overall fluorescence intensity of both melanin and lipofuscin granules clearly increased with increasing donor age, the increase being most marked for melanin. In all granule populations the fluorescence decays were multiexponential with subnanosecond and nanosecond decay components. The resultant time-integrated and time-gated spectra also exhibited marked age-variations for each type of granule. Young melanin showed spectral patterns similar to those of bovine melanin, while a yellow-orange fluorescence band appeared in melanin samples from older age groups. Lipofuscin granules exhibited a blue, a yellow and an orange band whose relative amounts were age-related. The results demonstrate the potential of time-resolved techniques for discriminating fluorophores in vitro and in situ, and have confirmed results previously obtained using extraction techniques. Furthermore, the ability of this technique to identify and quantify individual fluorophores within granules may provide an important insight into the origin and development of lipofuscin within the retinal pigment epithelium and ultimately into the mechanisms of age-related retinal diseases.     

Probing the spatial dependence of the emission spectrum of single human retinal lipofuscin granules using near-field scanning optical microscopy

The emission spectra of single lipofuscin granules are examined using spectrally resolved confocal microscopy and near-field scanning optical microscopy (NSOM). The emission spectrum varies among the granules examined revealing that individual granules are characterized by different distributions of fluorophores. The range of spectra observed is consistent with in vivo spectra of human retinal pigment epithelium cells. NSOM measurements reveal that the shape of the spectrum does not vary with position within the emissive regions of single lipofuscin granules. These results suggest that the relative distribution of fluorophores within the emissive regions of an individual granule is homogeneous on the spatial scale approximately 150 nm.     

Comparative study of the dark and light-induced toxicity of lipofuscin granules from human retinal pigment epithelium and their chromophore A2E on the cardiolipin liposome model

The damaging effect of lipofuscin granules from the human retinal pigment epithelium and fluorophore A2E was studied on models of calcein- and ascorbate-loaded cardiolipin liposomes and outer segments of the bovine eye photoreceptor cells in dark and under visible light irradiation. In dark fluorophore A2E induces the release of calcein from calcein-loaded liposomes and reduces the lifetime of the artificial bilayer lipid membrane prepared from dioleyl phosphatidilcholine. A similar detergent-like action A2E exhibits towards ascorbate-loaded liposomes, significantly accelerating the release of ascorbate in dark. In the presence of A2E, irradiation with the full visible light (390?C700 nm) stimulates both the release of ascorbate from liposomes and accelerates the destruction of the bilayer lipid membrane. Retinal pigment epithelium lipofuscin granules also accelerate the release of ascorbate from ascorbate-loaded liposomes under visible light irradiation; the blue light (457.9 nm) was twice as more efficient as the green light (514.5 nm). The preliminary irradiation of A2E with the visible light decreases its detergent-like action on the cardiolipin liposomal membranes under the dark conditions and the photosensitizing effect on the lipid peroxidation of the outer segments of photoreceptor cells. Unlike A2E, the visible light irradiation of a suspension of lipofuscin granules under similar conditions does not noticeably decrease their sensitizing activity towards lipid peroxidation. It is assumed that the phototoxicity of retinal pigment epithelium lipofuscin granules is related not only to A2E in their composition, but depends mainly on the content of other photosensitizers (chromophores) in the granules.     

Electron spin resonance investigations of human retinal pigment epithelium melanosomes from young and old donors

Electron spin resonance (ESR) examinations of human retinal pigment epithelium melanosomes isolated from eyes of young and old donors were carried out. The examined ESR signal was a single line, which is characteristic for free radicals of eumelanin o-semiquinones. The content of free radicals related to melanosomes dry weight for samples from older donors (ages over 45 years) were higher than for sample from younger donors (between 14 and 22 years). Simultaneously, the content of free radicals calculated for one melanosome is constant and does not depend on age. The homogeneous broadening of the recorded ESR lines shows that there are no isolated spin packets in all investigated melanin samples. Slow spin-lattice (T1 approximately 10(-5) s) and fast spin-spin (T2 approximately 10(-8) s) relaxation processes occur in these samples. Saturation of the ESR lines at low microwave power was measured. High concentration of free radicals in melanosome samples was responsible for the fast spin-spin relaxation process.     

Comparisons of the structural and chemical properties of melanosomes isolated from retinal pigment epithelium, iris and choroid of newborn and mature bovine eyes

Melanosomes were isolated from the retinal pigment epithelium (RPE), iris and choroid of mature (age >2 years) and newborn (age <1 week) bovine eyes. Scanning electron microscopy was utilized to analyze the morphology of the melanosomes, which were found to vary among different tissues and different ages. While the total content of amino acids differs slightly (ranging from 9% to 15% by mass), the distributions of the amino acids are similar. The pheomelanin content is low in the choroid and the RPE (0.1-0.5%), and moderate in the iris (<2%); therefore, the major melanin component of bovine eye melanosomes is eumelanin, independent of the shape of the melanosomes. The yields of pyrrole-2,3,5-tricarboxylic acid from melanosomes decrease in the following order: choroid > iris > RPE, and exhibit decreasing yields with age. 13C solid-state nuclear magnetic resonance (NMR) spectroscopic analysis of iris and choroid melanosomes indicates the same trends. These observations suggest that the 5,6-dihydroxyindole-2-carboxylic acid contents decrease in the following order: choroid > iris > RPE, and decrease with age. Moreover, the 13C solid-state NMR spectra show (1) for the same age samples, the CH:Cq ratio for choroid is larger than that for iris melanosomes; and (2) an increase in the concentration of carbonyl groups with age within each type of melanosome.     

Oxidation potentials of human eumelanosomes and pheomelanosomes

Eumelanosomes and pheomelanosomes isolated from black and red human hair, respectively, were studied by photoelectron emission microscopy (PEEM). PEEM images were collected at various wavelengths between 207 and 344 nm, using the spontaneous emission output of the Duke OK-4 free electron laser (FEL). Analysis of the FEL-PEEM data revealed ionization thresholds of 4.6 and 3.9 eV corresponding to oxidation potentials of -0.2 and +0.5 V vs normal hydrogen electrode for eumelanosomes and pheomelanosomes, respectively. The difference in oxidation potential is attributed to the pigment content of the melanosome, namely whether it contains primarily eumelanin and pheomelanin. The effect of added melanosomes on the reduction of Fe(III)-cytochrome showed pheomelanosomes are stronger reducing agents than eumelanosomes, consistent with the measured oxidation potentials. The FEL-PEEM experiment offers to be an important new approach for quantifying the effects of age, oxidation and metal accumulation on the oxidation potentials of intact melanosomes.     

Fluorophores of lipofuscin granules responsible for human eye fundus autofluorescence

Individual fluorophores and/or their groups contained in a chloroform extract of lipofuscin granules isolated from retinal pigment epithelium of human cadaver eyes were studied by HPLC. Their spectral characteristics were studied, which made it possible to evaluate the contribution of particular fluorophores and/or their groups to the general image of human eye fundus autofluorescence. Many components, being conjugates of all-trans-retinal of different nature, contribute to the total fluorescence spectrum of the chloroform extract. The fluorophore A2E is not predominant.     

Photobleaching of melanosomes from retinal pigment epithelium: II. Effects on the response of living cells to photic stress

Melanosomes of the retinal pigment epithelium (RPE) are long lived organelles that may undergo photobleaching with aging, which can diminish the antioxidant efficiency of melanin. Here, isolated porcine RPE melanosomes were experimentally photobleached with visible light to simulate aging and compared with untreated granules or control particles (black latex beads) for their effects on the survival of photically stressed ARPE-19 cultures. Particles were delivered to cultures for uptake by phagocytosis then cells were exposed to violet light and analyzed by a new live cell imaging method to identify the time of apoptotic blebbing as a dynamic measure of reduced cell survival. Results indicated that untreated melanosomes did not decrease photic injury to ARPE-19 cells when compared with cells lacking particles or with cells containing control particles, as might be expected if melanin performed an antioxidant function. Instead cells with untreated melanosomes showed reduced survival indicated by an earlier onset of blebbing and a lower fraction of surviving cells after photic stress. Cell survival was reduced even further in stressed cells containing melanosomes that were photobleached, and survival decreased with increasing photobleaching time. Photobleaching of RPE melanosomes therefore makes cells containing them more sensitive to light-induced cytotoxicity. This observation raises the possibility that aged melanosomes increase RPE cell photic stress in situ, perhaps contributing to reduced tissue function and to degeneration of the adjacent retina that the RPE supports. How melanosomes (photobleached or not) interact with their local subcellular environment to modify RPE cell survival is poorly understood and is likely determined by the physicochemical state of the granule and its constituent melanin. The live cell imaging method introduced here, which permitted detection of a graded effect of photobleaching, provides a sensitive bioassay for probing the effects of melanosome modifications.     

Photophysical Studies of A2-E, Putative Precursor of Lipofuscin, in Human Retinal Pigment Epithelial Cells

With age, human retinal pigment epithelial cells accumulate lipofuscin that can absorb photons in the visible range leading to light-induced damage and impaired vision. A putative precursor of lipofuscin, 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E, 5E,7E- octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1 - cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium (A2-E), has recently been isolated and characterized from aged human retinal pigment epithelial cells. We have found that A2-E inhibits the growth of human retinal pigment epithelial cells at concentrations greater than 1 microM. Time-resolved fluorescence measurements of 1 microM A2-E in solution, performed under 413 nm excitation, showed that fluorescence wave forms integrated across the spectrum (450-600 nm) were best-fitted with three decay times in the nanosecond and subnanosecond time scale: 6.6, 1.9 and 0.33 ns. Untreated retinal pigment epithelial cells were characterized by three fluorescence lifetimes: 6.3, 1.7 and 0.35 ns. In retinal pigment epithelial cells treated with 1 microM A2-E, the fluorescence decay was significantly faster, with the marked presence (approximately equal to 30%) of a fourth short lifetime (0.12 ns). These fluorescence decay times for A2-E bound to human retinal pigment epithelial cells are similar to those of lipofuscin granules isolated from aged human retinal pigment epithelial cells. This similarity supports the hypothesis that A2-E is a precursor of lipofuscin and suggests that A2-E may play a role in the overall light damage associated with age-related retinal diseases.     

Lipofuscin and A2E Accumulate with Age in the Retinal Pigment Epithelium of Nrl−/− Mice†

Lipofuscin is a fluorescent material with significant phototoxic potential that accumulates with age in the retinal pigment epithelium (RPE) of the eye. It is thought to be a factor in retinal degeneration diseases. The most extensively characterized lipofuscin component, N‐retinylidene‐N‐retinylethanolamine (A2E), has been proposed to be a byproduct of reactions involving the visual pigment chromophore. To examine the impact of the visual pigment and photoreceptor cell type on lipofuscin accumulation, we analyzed the RPE from Nrl^?/? mice of various ages for lipofuscin fluorescence and A2E levels. The photoreceptor cells of the Nrl^?/? retina contain only cone‐like pigments, and produce cone‐like responses to photostimulation. The cone‐like nature of these cells was confirmed by the presence of RPE65. Lipofuscin was measured with fluorescence imaging, whereas A2E was quantified by UV/VIS absorbance spectroscopy coupled to HPLC. The identity of A2E was corroborated with tandem mass spectrometry. Lipofuscin and A2E accumulated with age, albeit to lower levels compared with wild type mice. The emission spectra of RPE lipofuscin granules from Nrl^?/? mice were similar to those from wild type mice, with λ_maxca 610 nm. These results demonstrate that cone visual pigments can contribute to the production of lipofuscin and A2E.     

Photoionization thresholds of melanins obtained from free electron laser-photoelectron emission microscopy, femtosecond transient absorption spectroscopy and electron paramagnetic resonance measurements of oxygen photoconsumption

Free electron laser-photoelectron emission microscopy (FEL-PEEM), femtosecond absorption spectroscopy and electron paramagnetic resonance (EPR) measurements of oxygen photoconsumption were used to probe the threshold potential for ionization of eumelanosomes and pheomelanosomes isolated from human hair. FEL-PEEM data show that both pigments are characterized by an ionization threshold at 282 nm. However, pheomelanosomes exhibit a second ionization threshold at 326 nm, which is interpreted to be reflective of the benzothiazine structural motif present in pheomelanin and absent in eumelanin. The lower ionization threshold for pheomelanin is supported by femtosecond transient absorption spectroscopy. Unlike photolysis at 350 nm, following excitation of solubalized synthetic pheomelanin at 303 nm, the transient spectrum observed between 500 and 700 nm matches that for the solvated electron, indicating the photoionization threshold for the solubalized pigment is between 350 and 303 nm. For the same synthetic pheomelanin, EPR oximetry experiments reveal an increased rate of oxygen uptake between 338 nm and 323 nm, narrowing the threshold for photoionization to sit between these two wavelengths. These results on the solubalized synthetic pigment are consistent with the FEL-PEEM results on the human melanosomes. The lower ionization potential observed for pheomelanin could be an important part of the explanation for the greater incidence rate of UV-induced skin cancers in red-haired individuals.     

Properties and function of the ocular melanin--a photobiophysical view.

This paper reviews the biosynthesis and physicochemical properties of the ocular melanin. Age-related changes of melanin granules and the corresponding formation of lipofuscin pigments in the retinal pigment epithelium (RPE) are also described. Adverse photoreactions of the eye and, in particular, light-induced damage to the RPE-retina are reviewed in relation to the ocular pigmentation. A hypothesis on the photoprotective role of the RPE melanin is presented that is based on the ability of the cellular melanin to bind redoxactive metal ions. Since bound-to-melanin metal ions are expected to be less damaging to the pigment cells, it is proposed that sequestration of heavy metal ions by the RPE melanin is an efficient detoxifying mechanism. It is postulated that oxidative degradation of RPE melanin may lower its metal-binding capability and decrease its anti-oxidant efficiency. Cellular and environmental factors that may contribute to possible oxidative damage of the RPE melanin are discussed in connection with the etiology of age-related macular degeneration.     

Photobleaching of melanosomes from retinal pigment epithelium: I. Effects on protein oxidation

Melanin in the long-lived melanosomes of the retinal pigment epithelium (RPE) may undergo photobleaching with aging, which appears to diminish the antioxidant function of melanin and could make photobleached melanosomes less efficient in protecting biomolecules from oxidative modification. Here we analyzed whether photobleaching of melanosomes affects their ability to modify the oxidation state of nearby protein. As conventional methods developed to study soluble antioxidants are not well suited for analysis of granules such as melanosomes, we developed a new analytic method to focus on particle surfaces that involves experimentally coating granules with the cytoskeletal protein beta-actin to serve as a reporter for local protein oxidation. Isolated porcine RPE melanosomes were photobleached with visible light to simulate aging, then photobleached melanosomes, untreated melanosomes and control particles (black latex beads) were actin coated and illuminated in a photosensitized cell free system. Protein was re-stripped from particles and analyzed for carbonylation by Western blotting. Quantitative densitometry showed no reproducible differences for protein associated with untreated melanosomes when compared with control particles. Melanin has both anti- and pro-oxidant functions when light irradiated, but neither of these functions predominated in the protein oxidation assay when untreated melanosomes were used. However, protein extracted from photobleached melanosomes showed markedly increased carbonylation, both of associated actin and of endogenous melanosomal protein(s), and the effect increased with extent of granule photobleaching. Photobleaching of RPE melanosomes therefore changes the oxidation state of protein endogenous to the organelle and reduces the ability of the granule to modify the oxidation of exogenous protein near the particle surface. The results support the growing body of evidence that photobleaching of RPE melanosomes, which is believed to occur with aging, changes the physicochemical properties of the organelle and reduces the likelihood that the granules perform an antioxidant function.     

Fluorescence emission and excitation spectra of fluorophores of lipofuscin granules isolated from retinal pigment epithelium of human cadaver eyes

Visible light-induced changes in fluorescence characteristics of lipofuscin granules (LG) isolated from retinal pigment epithelium of human cadaver eyes are compared with the analogous age-related changes and correlated with the content and photooxidation of LG main fluorophore, A2E. We used HPLC to examine changes of LG fluorophore composition with donor age, as well as before and after visible-light irradiation (the latter HPLC tests were also done with synthetic A2E). Visible light induces oxidation of LG fluorophores. As a result, their fluorescence characteristics change: the emission spectrum is blue-shifted by 25–40 nm. The observed age-dependent changes in the relative content of LG fluorophores and their oxidized derivatives were qualitatively similar with those caused by irradiation. To improve the accuracy of a new noninvasive diagnostic method, fundus autofluorescence imaging, it is important to know the ratio of nonoxidized and oxidized fluorophore derivatives depending on age and eye pathology.     

PHOTOPHYSICAL STUDIES ON HUMAN RETINAL LIPOFUSCIN

Fluorescent material generated in the human retina accumulates within lipofuscin granules of the retinal pigment epithelium (RPE) during aging. Its presence has been suggested to contributed to various diseases including age-related macular degeneration. Because this material absorbs light at wave lengths as long as 550 nm, photophysical studies were performed to determine whether lipofuscin could contribute to light damage and to determine if its composition is similar to a synthetically prepared lipofuscin. Time-resolved experiments were performed to monitor (1) fluorescence decay, (2) the UV-visible absorption of longer-lived excited states and (3) the formation and decay of singlet oxygen at 1270 nm. Steady-state and time-resolved fluorescence studies indicate that human and synthetic lipofuscin have fluorophores in common. Time-resolved absorption experiments on human retinal lipofuscin and synthetic lipofuscin showed the presence of at least two transient species, one absorbing at 430 nm (lifetime caμs) and a second absorbing at 580 nm, which decays via second order kinetics. In addition, there is a third absorbing species stable to several hundred milliseconds. The transient species at 430 nm is quenched by oxygen, suggesting that it is a triplet state. Subsequent studies showed the formation of singlet oxygen, which was monitored by its phosphorescence decay at 1270 nm. These studies demonstrate that lipofuscin can act as a sensitizer for the generation of reactive oxygen species that may contribute to the age-related decline of RPE function and blue light damage.     

Neuromelanins Isolated from Different Regions of the Human Brain Exhibit a Common Surface Photoionization Threshold

Neuromelanin isolated from the premotor cortex, cerebellum, putamen, globus pallidus and corpus callosum of the human brain is studied by scanning probe and photoelectron emission microscopies and the results are compared with previously published work on neuromelanin from the substantia nigra. Scanning electron microscopy reveals common structure for all neuromelanins. All exhibit spherical entities of diameters between 200 and 400 nm, composed of smaller spherical substructures, ∼30 nm in diameter. These features are similar to that observed for many melanin systems including Sepia cuttlefish, bovine eye, and human eye and hair melanosomes. Photoelectron microscopy images were collected for all neuromelanins at specific wavelengths of ultraviolet light between 248 and 413 nm, using the spontaneous emission output from the Duke free electron laser. Analysis of the data establishes a common threshold photoionization potential for neuromelanins of 4.7 ± 0.2 eV, corresponding to an oxidation potential of −0.3 ± 0.2 V vs the normal hydrogen electrode (NHE). These results are consistent with previously reported potentials for neuromelanin from the substantia nigra of 4.5 ± 0.2 eV (−0.1 ± 0.2 V vs NHE). All neuromelanins exhibit a common low surface oxidation potential, reflecting their eumelanic component and their inability to trigger redox processes with neurotoxic effect.