Discriminant Principal Component Analysis of ToF-SIMS Spectra for Deciphering Compositional Differences of MSC-Secreted Extracellular Matrices.

Identifying characteristic extracellular matrix (ECM) variants is a key challenge in mechanistic biology, bioengineering, and medical diagnostics. The reported study demonstrates the potential of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to detect subtle differences between human mesenchymal stromal cell (MSC)-secreted ECM types as induced by exogenous stimulation or emerging pathology. ToF-SIMS spectra of decellularized ECM samples are evaluated by discriminant principal component analysis (DPCA), an advanced multivariate analysis technique, to decipher characteristic compositional features. To establish the approach, signatures of major ECM proteins are determined from samples of pre-defined mixtures. Based on that, sets of ECM variants produced by MSCs in vitro are analyzed. Differences in the content of collagen, fibronectin, and laminin in the ECM resulting from the combined supplementation of MSC cultures with polymers that induce macromolecular crowding and with ascorbic acid are detected from the DPCA of ToF-SIMS spectra. The results are verified by immunostaining. Finally, the comparative ToF-SIMS analysis of ECM produced by MSCs of healthy donors and patients suffering from myelodysplastic syndrome display the potential of the novel methodology to reveal disease-associated alterations of the ECM composition.

Trace metal sorption on nanoplastics: An innovative analytical approach combining surface analysis and mass spectrometry techniques.

The mass and volume concentration of nanoplastics is extremely low, but incredibly high in terms of surface area; this is expected to increase their toxicity through the ab/adsorption and transport of chemical co-pollutants such as trace metals. In this context, we studied the interactions between nanoplastics model materials functionalized with carboxylated groups, with either smooth or raspberry-like surface morphologies, and copper as representative of trace metals. For this purpose, a new methodology, using two complementary surface analysis techniques: Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and X-ray Photoelectron Spectroscopy (XPS) was developed. In addition, inductively coupled plasma mass spectrometry (ICP-MS) was used to quantify the total mass of sorbed metal on the nanoplastics. This innovative analytical approach from the top surface to the core of nanoplastics demonstrated not only the interactions with copper at the surface level, but also the ability of nanoplastics to absorb metal at their core. Indeed, after 24 h of exposition, the copper concentration at the nanoplastic surface remained constant due to saturation whereas the copper concentration inside the nanoplastic keeps increasing with the time. The sorption kinetic was evaluated to increase with the density of charge of the nanoplastic and the pH. This study confirmed the ability of nanoplastics to act as metal pollutant carriers by both adsorption and absorption phenomena.

240Pu/239Pu isotopic ratio measurements in micrometric Pu and MOX particles using Secondary Ion Mass Spectrometry.

Every accident affecting industrial or nuclear facilities emits micrometric fragments of material into the environment whose elemental and isotopic compositions are characteristic of the process or event. Particle analysis, mainly implemented in the framework of the Non Proliferation Treaty to detect clandestine nuclear activities, provides a powerful tool to identify the origin of the nuclear particulate matter and to assess the environmental impact of nuclear accidents. Initially, particle-scale isotopic analyses aimed at the determination of the U isotopic composition. Now, focus is increasingly given on Pu isotopic measurements to address its origin and potential use. Such measurements are more challenging because of isobaric interferences, including those induced by hydride ions, like 239PuH+ on 240Pu+ and 238UH+ on 239Pu+ in Mixed Oxide (MOX). Such ions are generated during ionization processes by Secondary Ion Mass Spectrometry. Based on a parametric study aiming at the measurement of uranium oxide, uranium carbide and uranium single and double hydride rates, we determined that Pu and U should be detected as elementary ions to limit the impact of such interferences, although mono-oxide ions are more abundant. Thus, we developed an analytical methodology to obtain accurate 240Pu/239Pu atomic ratios both for weapon grade Pu and MOX materials. Hydride rate is first measured in U oxide particles and then applied to correct 240Pu+ and 239Pu+ signals. The relative difference of corrected 240Pu/239Pu isotopic ratios with expected values is reduced by a factor of 4 when measuring weapon grade Pu particles and by a factor of 10-100 when measuring MOX particles containing 1 to 10 wt% of Pu. We also proposed a method to determine the Relative Sensitivity Factor (RSF) based on the decay of Pu in order to quantify the Pu content in MOX samples. The estimated lowest measurable 239Pu/238U atomic ratio in MOX particles is ∼1.6 × 10-3.

Development of Mercury Analysis by NanoSIMS for the Localization of Mercury-Selenium Particles in Whale Liver.

Nanoscale secondary ion mass spectrometry (NanoSIMS) is a dynamic SIMS technique, which offers high spatial resolution allowing the mapping of chemical elements at the nanometer scale combined with high sensitivity. However, SIMS for mercury analysis is a challenging issue due to the low secondary ion yield and has never been done on NanoSIMS. The introduction of an rf plasma oxygen primary ion source on NanoSIMS enabled higher lateral resolution and higher sensitivity for electropositive elements such as most metals. In this paper, for the first time, mercury analysis by NanoSIMS was developed applying the new rf plasma O- ion source. All mercury isotopes could be detected as Hg+ secondary ions and the isotopic pattern corresponded to their natural isotopic abundances. Furthermore, Hg+ detection in HgSe nanocrystals has been investigated where polyatomic interferences from selenium clusters were identified and separated by high mass resolution (ΔM/M ≥ 3200). However, in the presence of selenium a strong matrix effect was observed, decreasing the Hg+ secondary ion yield. In addition, a detection of Se+ ions was possible, too. The newly developed method was successfully applied to nanoscale localization by chemical imaging of HgSe particles accumulated in the liver tissue of sperm whale (Physeter macrocephalus). This demonstrated the applicability of NanoSIMS not only for mercury detection in surface analysis but also for mercury mapping in biological samples.

Localization of (+)-Catechin in Picea abies Phloem: Responses to Wounding and Fungal Inoculation.

To understand the positional and temporal defense mechanisms of coniferous tree bark at the tissue and cellular levels, the phloem topochemistry and structural properties were examined after artificially induced bark defense reactions. Wounding and fungal inoculation with Endoconidiophora polonica of spruce bark were carried out, and phloem tissues were frequently collected to follow the temporal and spatial progress of chemical and structural responses. The changes in (+)-catechin, (-)-epicatechin, stilbene glucoside, and resin acid distribution, and accumulation patterns within the phloem, were mapped using time-of-flight secondary ion mass spectrometry (cryo-ToF-SIMS), alongside detailed structural (LM, TEM, SEM) and quantitative chemical microanalyses of the tissues. Our results show that axial phloem parenchyma cells of Norway spruce contain (+)-catechins, the amount of which locally increases in response to fungal inoculation. The preformed, constitutive distribution and accumulation patterns of (+)-catechins closely follow those of stilbene glucosides. Phloem phenolics are not translocated but form a layered defense barrier with oleoresin compounds in response to pathogen attack. Our results suggest that axial phloem parenchyma cells are the primary location for (+)-catechin storage and synthesis in Norway spruce phloem. Chemical mapping of bark defensive metabolites by cryo-ToF-SIMS, in addition to structural and chemical microanalyses of the defense reactions, can provide novel information on the local amplitudes and localizations of chemical and structural defense mechanisms and pathogen-host interactions of trees.

Mechanisms of lipid preservation in archaeological clay ceramics revealed by mass spectrometry imaging.

Traces of lipids, absorbed and preserved for millennia within the inorganic matrix of ceramic vessels, act as molecular fossils and provide manifold information about past people's subsistence, diet, and rituals. It is widely assumed that lipids become preserved after adsorption into nano- to micrometer-sized pores, but to this day the distribution of these lipids in the ceramics was virtually unknown, which severely limits our understanding about the process of lipid preservation. Here we use secondary ion mass spectrometry (SIMS) imaging for direct in situ analysis of lipids absorbed in 700- to 2,000-y-old archaeological pottery. After sectioning from larger sherds, wall cross-sections of smaller fragments were used for SIMS analysis. Lipids were found in relatively large zones of 5- to 400-µm diameter, which does not support the notion of absorption only into individual nanometer-scale pores but indicates that more macroscopic structures in the ceramics are involved in lipid preservation as well. Furthermore, lipids were found concentrated on calcium carbonate inclusions in the ceramics, which suggests that precipitation of fatty acids as calcium salts is an important aspect of lipid preservation in archaeological samples. This has important implications for analytical methods based on extraction of lipids from archaeological ceramics and needs to be considered to maximize the yield and available information from each unique sample.

Zinc Deficiency Leads to Lipid Changes in Drosophila Brain Similar to Cognitive-Impairing Drugs: An Imaging Mass Spectrometry Study.

Several diseases and disorders have been suggested to be associated with zinc deficiency, especially learning and memory impairment. To have better understanding about the connection between lipid changes and cognitive impairments, we investigated the effects of a zinc-chelated diet on certain brain lipids of Drosophila melanogaster by using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The data revealed that there are increases in the levels of phosphatidylcholine and phosphatidylinositol in the central brains of the zinc-deficient flies compared to the control flies. In contrast, the abundance of phosphatidylethanolamine in the brains of the zinc-deficient flies is lower. These data are consistent with that of cognitive-diminishing drugs, thus providing insight into the biological and molecular effects of zinc deficiency on the major brain lipids and opening a new treatment target for cognitive deficit in zinc deficiency.

Effect of sample preparation techniques upon single cell chemical imaging: A practical comparison between synchrotron radiation based X-ray fluorescence (SR-XRF) and Nanoscopic Secondary Ion Mass Spectrometry (nano-SIMS).

Analytical capabilities of Nanoscopic Secondary Ion Mass Spectrometry (nano-SIMS) and Synchrotron Radiation based X-ray Fluorescence (SR nano-XRF) techniques were compared for nanochemical imaging of polymorphonuclear human neutrophils (PMNs). PMNs were high pressure frozen (HPF), cryo-substituted, embedded in Spurr's resin and cut in thin sections (500 nm and 2 µm for both techniques resp.) Nano-SIMS enabled nanoscale mapping of isotopes of C, N, O, P and S, while SR based nano-XRF enabled trace level imaging of metals like Ca, Mn, Fe, Ni, Cu and Zn at a resolution of approx. 50 nm. The obtained elemental distributions were compared with those of whole, cryofrozen PMNs measured at the newly developed ID16A nano-imaging beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Similarities were observed for elements more tightly bound to the cell structure such as phosphorus and sulphur, while differences for mobile ions such as chlorine and potassium were more pronounced. Due to the observed elemental redistribution of mobile ions such as potassium and chlorine, elemental analysis of high pressure frozen (HPF), cryo-substituted and imbedded cells should be interpreted critically. Although decreasing analytical sensitivity occurs due to the presence of ice, analysis of cryofrozen cells - close to their native state - remains the golden standard. In general, we found nanoscale secondary ion mass spectrometry (nano-SIMS) and synchrotron radiation based nanoscopic X-ray fluorescence (SR nano-XRF) to be two supplementary alternatives for nanochemical imaging of single cells at the nanoscale.

The Vitamin A and D Exposure of Cells Affects the Intracellular Uptake of Aluminum Nanomaterials and its Agglomeration Behavior: A Chemo-Analytic Investigation.

Aluminum (Al) is extensively used for the production of different consumer products, agents, as well as pharmaceuticals. Studies that demonstrate neurotoxicity and a possible link to Alzheimer's disease trigger concern about potential health risks due to high Al intake. Al in cosmetic products raises the question whether a possible interaction between Al and retinol (vitamin A) and cholecalciferol (vitamin D3) metabolism might exist. Understanding the uptake mechanisms of ionic or elemental Al and Al nanomaterials (Al NMs) in combination with bioactive substances are important for the assessment of possible health risk associated. Therefore, we studied the uptake and distribution of Al oxide (Al2O3) and metallic Al0 NMs in the human keratinocyte cell line HaCaT. Possible alterations of the metabolic pattern upon application of the two Al species together with vitamin A or D3 were investigated. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging and inductively coupled plasma mass spectrometry (ICP-MS) were applied to quantify the cellular uptake of Al NMs.

ToF-SIMS 3D imaging unveils important insights on the cellular microenvironment during biomineralization of gold nanostructures.

The biomolecular imaging of cell-nanoparticle (NP) interactions using time-of-flight secondary ion mass spectrometry (ToF-SIMS) represents an evolving tool in nanotoxicology. In this study we present the three dimensional (3D) distribution of nanomaterials within biomolecular agglomerates using ToF-SIMS imaging. This novel approach was used to model the resistance of human alveolar A549 cells against gold (Au) ion toxicity through intra- and extracellular biomineralization. At low Au concentrations (≤1 mM HAuCl4) 3D-ToF-SIMS imaging reveals a homogenous intracellular distribution of Au-NPs in combination with polydisperse spherical NPs biomineralized in different layers on the cell surface. However, at higher precursor concentrations (≥2 mM) supplemented with biogenic spherical NPs as seeds, cells start to biosynthesize partially embedded long aspect ratio fiber-like Au nanostructures. Most interestingly, A549 cells seem to be able to sense the enhanced Au concentration. They change the chemical composition of the extracellular NP agglomerates from threonine-O-3-phosphate aureate to an arginine-Au(I)-imine. Furthermore they adopt the extracellular mineralization process from spheres to irregular structures to nanoribbons in a dose-dependent manner with increasing Au concentrations. The results achieved regarding size, shape and chemical specificity were cross checked by SEM-EDX and single particle (sp-)ICP-MS. Our findings demonstrate the potential of ToF-SIMS 3D imaging to better understand cell-NP interactions and their impact in nanotoxicology.

ToF-SIMS spectrometry to observe fatty acid profiles of breast tissues in broiler chicken subjected to varied vegetable oil diet.

This study is aimed to observe changes in fatty acid profiles by time of flight secondary ion mass spectrometry (ToF-SIMS) in breast muscle tissues of broilers. Four different groups were identified. The source of fat in group I was soy oil (rich in linoleic acid, ω-6), group II received linseed oil (ω-3), and the third group was fed a mixture of the two mentioned oils. Broilers in the control group were fed with beef tallow, used in mass commercial production. The results reveal that the use of vegetable oils in animal nutrition determines the lipid profile of fatty acids. ToF-SIMS measurements showed that the lipid profile of muscle fibers and intramuscular fat reflect the composition of fats used as feed additives. In both structures, the ratio of ω-6/ω-3 fatty acids, which is most favorable for human health, was found in the groups in which a mixture of vegetable oils and a supplement of linseed oil were used.

Investigation of architecture development and phosphate distribution in Chlorella biofilm by complementary microscopy techniques.

Microalgae biofilms may play an important role in the mitigation and prevention of eutrophication caused by domestic, agricultural and industrial wastewater effluents. Despite their potential, the biofilm development and role in nutrient removal are not well understood. Its clarification requires comprehensive studies of the complex three-dimensional architecture of the biofilm. In this study, we established a multimodal imaging approach to provide key information regarding architecture development and nutrient distribution in the biofilm of two green algae organisms: Chlorella pyrenoidosa and Chlorella vulgaris. Helium ion microscopy (HIM), scanning electron microscopy coupled with energy dispersive X-ray analysis (SEM-EDX) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were employed for i) elucidation of spatial arrangement, ii) elemental mapping and iii) 3D chemical imaging of the biofilm. The fine structure of the algal biofilm was resolved by HIM, the evidence of the accumulation of phosphate in hot spots was provided by SEM-EDX and the localization of phosphate oxides granules throughout the whole sample was clarified by ToF-SIMS. The reported results shed light on the phosphorus distribution during Chlorella's biofilm formation and highlight the potential of such correlative approach to solve fundamental question in algal biotechnology research.

Imaging of vitamin D in psoriatic skin using time-of-flight secondary ion mass spectrometry (ToF-SIMS): A pilot case study.

Recently it has been recognized that there is a need of investigating in detail the vitamin D synthesis and metabolism directly in the skin with respect to its possible autocrine and paracrine actions. The potential effects the active metabolite of vitamin D exerts in pathological skin conditions like psoriasis needs to be clarified. Under ultraviolet B (UVB) irradiation skin can autonomously synthesize, activate and degrade vitamin D. In this pilot case study, we used for the first time Time-of-flight secondary ion mass spectrometry (ToF-SIMS) in the analysis of skin biopsies from a patient with psoriasis before and after UVB phototherapy. We were able to visualize vitamin D3 and its metabolites in the skin and subcutaneous tissue. At the same time information about their localization at subcellular level and morphology of the skin was received. This study proves that ToF-SIMS is a promising powerful tool to be used when investigating vitamin D´s role in dermatological diseases through skin biopsies.

Single-cell imaging of phosphorus uptake shows that key harmful algae rely on different phosphorus sources for growth.

Single-cell measurements of biochemical processes have advanced our understanding of cellular physiology in individual microbes and microbial populations. Due to methodological limitations, little is known about single-cell phosphorus (P) uptake and its importance for microbial growth within mixed field populations. Here, we developed a nanometer-scale secondary ion mass spectrometry (nanoSIMS)-based approach to quantify single-cell P uptake in combination with cellular CO2 and N2 fixation. Applying this approach during a harmful algal bloom (HAB), we found that the toxin-producer Nodularia almost exclusively used phosphate for growth at very low phosphate concentrations in the Baltic Sea. In contrast, the non-toxic Aphanizomenon acquired only 15% of its cellular P-demand from phosphate and ~85% from organic P. When phosphate concentrations were raised, Nodularia thrived indicating that this toxin-producer directly benefits from phosphate inputs. The phosphate availability in the Baltic Sea is projected to rise and therefore might foster more frequent and intense Nodularia blooms with a concomitant rise in the overall toxicity of HABs in the Baltic Sea. With a projected increase in HABs worldwide, the capability to use organic P may be a critical factor that not only determines the microbial community structure, but the overall harmfulness and associated costs of algal blooms.

Imaging the distribution of skin lipids and topically applied compounds in human skin using mass spectrometry.

The barrier functions of skin against water loss, microbial invasion and penetration of xenobiotics rely, in part, on the spatial distribution of the biomolecular constituents in the skin structure, particularly its horny layer (stratum corneum). However, all skin layers are important to describe normal and dysfunctional skin conditions, and to develop adapted therapies or skin care products. In this work, time-of-flight secondary ion mass spectrometry (ToF-SIMS) combined with scanning electron microscopy (SEM) was used to image the spatial distribution of a variety of molecular species, from stratum corneum down to dermis, in cross-section samples of human abdominal skin. The results demonstrate the expected localization of ceramide and saturated long-chain fatty acids in stratum corneum (SC) and cholesterol sulfate in the upper part of the viable epidermis. The localization of exogenous compounds is demonstrated by the detection and imaging of carvacrol (a constituent of oregano or thyme essential oil) and ceramide, after topical application onto ex vivo human skin. Carvacrol showed pronounced accumulation to triglyceride-containing structures in the deeper parts of dermis. In contrast, the exogenous ceramide was found to be localized in SC. Furthermore, the complementary character of this approach with classical ex vivo skin absorption analysis methods is demonstrated.

New SIMS U-Pb age constraints on the largest lake transgression event in the Songliao Basin, NE China.

The largest lake transgression event (LTE) associated with lake anoxic events (LAE) and periodic seawater incursion events (SWIE) in the Songliao Basin, northeastern China, occurred during deposition of the Cretaceous Nenjiang Formation. The Yaojia-Nenjiang Formation boundary (YNB) marks the beginning of the LTE, as well as LAE and SWIE. However, there is an absence of direct radioisotopic dating, and therefore the age of the YNB, as well as the beginning of LTE, together with their relationship with other geological events, is strongly debated. Here we present a new SIMS U-Pb zircon age from the lowermost Nenjiang Formation. The bentonite bed located 9.88 m above the YNB of the X1-4 borehole was analyzed. Twenty-five analyses of 25 zircons were conducted, which produced a weighted mean age of 85.5±0.6 Ma (MSWD = 0.87). Based on the average sediment accumulation rate, the age of the YNB is suggested to be 85.7 Ma, indicating that the LTE began in the Early Santonian. The new ages provide a precise chronostratigraphic framework for climatic and geological events. Our new results imply that the beginning of the LTE, LAE and SWIE occurred almost simultaneously with short-term sea level rise, and probably had a close relationship with OAE3.

Intracellular uranium distribution: Comparison of cryogenic fixation versus chemical fixation methods for SIMS analysis.

Localization of uranium within cells is mandatory for the comprehension of its cellular mechanism of toxicity. Secondary Ion Mass Spectrometry (SIMS) has recently shown its interest to detect and localize uranium at very low levels within the cells. This technique requires a specific sample preparation similar to the one used for Transmission Electronic Microscopy, achieved by implementing different chemical treatments to preserve as much as possible the living configuration uranium distribution into the observed sample. This study aims to compare the bioaccumulation sites of uranium within liver or kidney cells after chemical fixation and cryomethods preparations of the samples: SIMS analysis of theses samples show the localization of uranium soluble forms in the cell cytoplasm and nucleus with a more homogenous distribution when using cryopreparation probably due to the diffusible portion of uranium inside the cytoplasm.

Interrogating marine virus-host interactions and elemental transfer with BONCAT and nanoSIMS-based methods.

While the collective impact of marine viruses has become more apparent over the last decade, a deeper understanding of virus-host dynamics and the role of viruses in nutrient cycling would benefit from direct observations at the single-virus level. We describe two new complementary approaches - stable isotope probing coupled with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence-based biorthogonal non-canonical amino acid tagging (BONCAT) - for studying the activity and biogeochemical influence of marine viruses. These tools were developed and tested using several ecologically relevant model systems (Emiliania huxleyi/EhV207, Synechococcus sp. WH8101/Syn1 and Escherichia coli/T7). By resolving carbon and nitrogen enrichment in viral particles, we demonstrate the power of nanoSIMS tracer experiments in obtaining quantitative estimates for the total number of viruses produced directly from a particular production pathway (by isotopically labelling host substrates). Additionally, we show through laboratory experiments and a pilot field study that BONCAT can be used to directly quantify viral production (via epifluorescence microscopy) with minor sample manipulation and no dependency on conversion factors. This technique can also be used to detect newly synthesized viral proteins. Together these tools will help fill critical gaps in our understanding of the biogeochemical impact of viruses in the ocean.

Identification of Distinct Functional Microstructural Domains Controlling C Storage in Soil.

The physical, chemical, and biological processes forming the backbone of important soil functions (e.g., carbon sequestration, nutrient and contaminant storage, and water transport) take place at reactive interfaces of soil particles and pores. The accessibility of these interfaces is determined by the spatial arrangement of the solid mineral and organic soil components, and the resulting pore system. Despite the development and application of novel imaging techniques operating at the micrometer and even nanometer scale, the microstructure of soils is still considered as a random arrangement of mineral and organic components. Using nanoscale secondary ion mass spectroscopy (NanoSIMS) and a novel digital image processing routine adapted from remote sensing (consisting of image preprocessing, endmember extraction, and a supervised classification), we extensively analyzed the spatial distribution of secondary ions that are characteristic of mineral and organic soil components on the submicrometer scale in an intact soil aggregate (40 measurements, each covering an area of 30 µm × 30 µm with a lateral resolution of 100 nm × 100 nm). We were surprised that the 40 spatially independent measurements clustered in just two complementary types of micrometer-sized domains. Each domain is characterized by a microarchitecture built of a definite mineral assemblage with various organic matter forms and a specific pore system, each fulfilling different functions in soil. Our results demonstrate that these microarchitectures form due to self-organization of the manifold mineral and organic soil components to distinct mineral assemblages, which are in turn stabilized by biophysical feedback mechanisms acting through pore characteristics and microbial accessibility. These microdomains are the smallest units in soil that fulfill specific functionalities.

Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model.

Purpose: Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Methods: Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Results: Apparently functional primary RPE cells, when cultured on 10-µm-thick inserts with 0.4-µm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. Conclusions: The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.