Optical Microspectroscopy Research Articles

Measurements of Spontaneous and External Stimuli Molecular Release Processes from a Single Optically Trapped Poly(lactic-co-glycolic) Acid Microparticle and a Liposome Containing Gold Nanospheres.

We investigated the single particle kinetics of the molecular release processes from two types of microcapsules used as drug delivery systems (DDS): biodegradable poly(lactic-co-glycolic) acid (PLGA) and a light-triggered-degradable liposome encapsulating gold nanospheres (liposome-GNP). To optimize the design of DDS capsules, it is highly desirable to develop a method for real-time monitoring of the release process. Using a combination of optical tweezers and confocal fluorescence microspectroscopy we successfully analyzed a single optically trapped PLGA particle and liposome-GNPs in solution. From temporal decay profiles of the fluorescence intensity, we determined the time constant τ of the release processes. We demonstrated that the release rate of spontaneously degradable microcapsules (PLGA) decreased with increasing size, while conversely, the release rate of external stimuli-degradable microcapsules (liposome-GNPs) increased in proportion to their size. This result is explained by the differences in the disruption mechanisms of the capsules, with PLGA undergoing hydrolysis and the GNPs in the liposome-GNP undergoing a photoacoustic effect under nanosecond pulsed laser irradiation. The present approach offers a way forward to an alternative microanalysis system for single drug delivery nanocarriers.

Spectral histology of hair and hair follicle using infrared microspectroscopy.

Today, there is only limited knowledge of the spatial organization of hair chemistry. Infrared microspectroscopy is a well-established tool to provide such information and has significantly contributed to this field. In this study, we present new results combining multiple infrared microspectroscopy methods at different length scales to create a better chemical histology of human hair, including the hair follicle, hair shaft, hair medulla and hair cuticle. We used hyperspectral IR imaging & spectroscopy (HIRIS) and synchrotron-radiation FTIR microspectroscopy (SR-μFTIR) to measure transversal hair sections and SR-μFTIR to obtain high-resolution maps of longitudinal sections from the hair shaft and from the hair follicle. We used optical photothermal IR microspectroscopy (OPTIR) to analyse the cuticle surface of intact hairs. By mapping longitudinal sections of the human hair follicle with confocal SR-μFTIR, we report the first demonstration of glycogen presence in the outer root sheath of the hair follicle by spectroscopy, and its quantification at the micron scale. Spectral maps, combined with machine learning-based analysis, enabled us to differentiate the various layers of the hair follicle and provided insights into the chemical changes that occur during hair formation in the follicle. Using HIRIS and SR-μFTIR to analyse the hair medulla in transversal sections of human hairs, we report here, for the first time by vibrational spectroscopy methods, the detection of unsaturated lipids at very low concentrations in the medulla. By analysing longitudinal sections of the hair shaft with SR-μFTIR, we found that calcium carboxylates are present in large regions of the hair cuticle, and not just in small focal areas as previously thought. We then use OPTIR to analyse the hair cuticle of intact hairs at submicron resolution without sectioning and report the distribution of calcium carboxylates at the surface of intact hair for the first time. These new findings illustrate the potential of infrared microspectroscopy for imaging the chemical composition of human hair and may have implications for biomedical research or cosmetology.

Identification of Microplastics Using µ-Raman Spectroscopy in Surface and Groundwater Bodies of SE Attica, Greece

Sixteen surface (5) and groundwater (11) samples were collected from the south-eastern part of Attica, Greece, and analysed for physico-chemical parameters and microplastics (MPs) by optical microscopy and Raman microspectroscopy (RS). A total of 3399 particles were optically identified in all sixteen samples, ranging from only 16 particles/L in a sample from a deeper borehole to 513 particles/L in a sample from a shallow water well. They were then visually classified into eight categories based on their color, texture, size, reflectivity, shape, and general morphological properties. Raman microspectroscopy was performed on the particles on the filters and revealed four different types of MPs, namely polyethylene (PE, 35%), polypropylene (PP, 30%), polystyrene (PS, 10%), and polyethylene terephthalate (PET, 25%). The samples from the shallow phreatic aquifer contained more MPs than the samples from the deeper borehole, which contained fewer MPs and categories. This is to be expected, since the phreatic aquifer (a) is generally more contaminated, as it is close to human activities that generate MPs and its infiltration depth is only a few metres, which means that many microplastics can infiltrate at such shallow depths, and (b) it is exposed to the atmosphere, so they can also be suspended in the air. On the other hand, it is interesting to note that MPs, especially PET and PE, were detected in the borehole sample, suggesting that MPs can migrate to greater depths through water infiltration. Chemical analyses of the groundwater samples revealed high values of E.C., Ca2+, Mg2+, Cl−, and Na+, which indicate that seawater intrusion is taking place in the coastal aquifer system of the Erasinos basin. The increased concentrations of NO3− and PO43− also indicate the impact of agricultural activities.

Hyperspectral photoluminescence and reflectance microscopy of 2D materials

Optical micro-spectroscopy is an invaluable tool for studying and characterizing samples ranging from classical semiconductors to low-dimensional materials and heterostructures. To date, most implementations are based on point-scanning techniques, which are flexible and reliable, but slow. Here, we describe a setup for highly parallel acquisition of hyperspectral reflection and photoluminescence (PL) microscope images using a push-broom technique. Spatial as well as spectral distortions are characterized and their digital corrections are presented. We demonstrate close- to diffraction-limited spatial imaging performance and a spectral resolution limited by the spectrograph. The capabilities of the setup are demonstrated by recording a hyperspectral PL map of a MoSe2–WSe2 lateral heterostructure, grown by chemical vapor deposition (CVD), from which we extract the luminescence energies, intensities and peak widths across the interface.

Real-time optical monitoring of non-isothermal bleaching of γ-irradiated International Simple Glass (ISG)

The thermal bleaching of γ-irradiated (0.83 and 1.99 MGy) nuclear waste glass simulant known as International Simple Glass (ISG, made by Mo-SCI Corporation, Rolla, MO) was studied in real time by non-isothermal in situ UV/Vis optical microspectroscopy. The method follows the Urbach energy temperature/time evolution for three constant heating rates. A novel opto-thermal technique for kinetic analysis, coined temperature scanning bleaching spectroscopy (TSBS) is proposed. The applicability of the Kissinger equation is extended by synergistic inflection point analysis of the Urbach energy time/temperature sigmoid. The sigmoidal behavior is discussed in the context of polaron time/temperature trends of the main factors of thermally generated polaron flux. The overall activation energy of the non-isothermal thermal bleaching is evaluated at 1.6 (±0.1) and 1.3 (±0.2) eV, for 0.83 and 1.99 MGy, respectively. The proposed TSBS methodology is applicable for real-time monitoring and robust quantitative comparison of non-isothermal thermal bleaching of any glassy material.

In Situ Optical Microspectroscopy Study of Isothermal Bleaching of γ-Irradiated International Simple Glass.

The isothermal bleaching of γ-irradiated glass was studied at elevated temperatures (280-340 °C) by real-time in situ optical microspectroscopy for the first time. The study was performed on γ-irradiated (0.83 and 1.99 MGy) International Simple Glass (ISG) borosilicate nuclear waste simulant made by Mo-SCI Corporation (Rolla, MO, USA). The current investigation proposes real-time optical transmission methodology for the activation energy assessment of isothermal bleaching of γ-irradiated glass. The method is based on robust quantification of the Urbach energy decay rates and yields similar activation energies for both doses within ∼0.24-0.26 eV.

Advanced Mapping of Optically-Blind and Optically-Active Nitrogen Chemical Impurities in Natural Diamonds

Natural diamonds with a rich variety of optically blind and optically active nitrogen impurity centers were explored at a nano/microscale on the surface and in bulk by a number of advanced chemical and structural analytical tools in order to achieve a comprehensive characterization by establishing enlightening links between their analysis results. First, novel compositional relationships were established between high-energy X-ray photoelectron spectroscopy (XPS) and low-energy Fourier-transform infrared vibrational spectroscopy (FT-IR) signals of nitrogen impurity defects acquired in the microscopy mode at the same positions of the diamond surface, indicating the verification XPS modality for qualitative and quantitative FT-IR analysis of high concentrations of nitrogen and other chemical impurity defects in diamond. Second, depth-dependent spatial distributions of diverse photoluminescence (PL)-active nitrogen defects were acquired in the confocal scanning mode in an octahedral diamond and then for the first time corrected to the related Raman signals of the carbon lattice to rule out artefacts of the confocal parameter and to reveal different micron-scale ontogenetic layers in the impurity distributions on its surface. Third, intriguing connections between local structural micro-scale defects (dislocation slip bands of plastic deformation zones) visualized by optical microscopy and Raman microspectroscopy, and related distributions of stress-sensitive PL-active nitrogen impurity defects in the proximity of these planes inside bulk diamonds were revealed. These findings demonstrate the broad instrumental opportunities for comprehensive in situ studies of the chemical, structural, and mechanical micro-features in diamonds, from the surface into bulk.

Permanent optical bleaching in HPHT-diamond via aggregation of C- and NV-centers excited by visible-range femtosecond laser pulses

Red-colored high-pressure, high-temperature synthesized (HPHT) diamond was exposed in its bulk in a number of patterns to millions of 0.3-ps, 515-nm laser pulses focused by a 0.25-NA micro-objective. The resulting arrays of bright micrometer-scale spots produced at variable laser exposure and pulse energy was characterized by optical transmission microspectroscopy, room-temperature dynamic and stationary 3D confocal photoluminescence (PL) microspectroscopy, FT-IR microspectroscopy. These bleached spots exhibit 1) reduced IR-absorbance of C- and A-centers at the pertaining absorbance of interstitials, 2) stronger reduced visible-range absorbance of C centers, comparing to that of NV0 and NV− centers, 3) considerably depth-wise reduced PL intensity of NV0 and NV− centers in favor of the emerging blue-green (480–550 nm) PL band of indistinguishable higher-aggregated H3, H4 centers. The photo-activated aggregation process of nitrogen centers exhibits second-power dependence on laser energy and is apparently related to direct agitation of low-aggregated centers, rather than to photo-generation of interstitial-vacancy pairs.

Optical-facilitated single-entity electrochemistry

Single-entity electrochemistry focusing on the study of transient electrochemical process at the confined interface, has become a promising field that addresses questions from multi-disciplines such as cellular biology, material chemistry, organic chemistry, etc . It offers the fruitful information hidden in bulk electrochemical measurements. As the optical techniques improve in spatial and temporal resolution, the combination of electrochemistry with optical microspectroscopy provides more comprehensive information of single-entity electrochemistry. Herein, we review recent progress made in optical–electrochemical measurements covering three aspects from the precise localization and temperature measurements of single compartments, to the in-situ tracking of dynamic behaviors of single nanoparticles in electrochemical process, and to the monitoring confinement-controlled electrochemistry at the single molecule/ion level. The review demonstrates how these optical methods are innovatively integrated with single-entity sensing. It also reveals how these optical–electrochemical combinations push single-entity electrochemistry forward.

Design and Validation of Nanofibers Made of Self-Assembled Peptides to Become Multifunctional Stimuli-Sensitive Nanovectors of Anticancer Drug Doxorubicin.

Self-assembled peptides possess remarkable potential as targeted drug delivery systems and key applications dwell anti-cancer therapy. Peptides can self-assemble into nanostructures of diverse sizes and shapes in response to changing environmental conditions (pH, temperature, ionic strength). Herein, we investigated the development of self-assembled peptide-based nanofibers (NFs) with the inclusion of a cell-penetrating peptide (namely gH625) and a matrix metalloproteinase-9 (MMP-9) responsive sequence, which proved to enhance respectively the penetration and tumor-triggered cleavage to release Doxorubicin in Triple Negative Breast Cancer cells where MMP-9 levels are elevated. The NFs formulation has been optimized via critical micelle concentration measurements, fluorescence, and circular dichroism. The final nanovectors were characterized for morphology (TEM), size (hydrodynamic diameter), and surface charge (zeta potential). The Doxo loading and release kinetics were studied in situ, by optical microspectroscopy (fluorescence and surface-enhanced Raman scattering–SERS). Confocal spectral imaging of the Doxo fluorescence was used to study the TNBC models in vitro, in cells with various MMP-9 levels, the drug delivery to cells as well as the resulting cytotoxicity profiles. The results confirm that these NFs are a promising platform to develop novel nanovectors of Doxo, namely in the framework of TNBC treatment.

Combined in vitro and in vivo investigation of barite microcrystals in Spirogyra (Zygnematophyceae, Charophyta)

We have investigated the biomineralisation of barite ‒a useful proxy for reconstructing paleoproductivity‒ in a freshwater alga, Spirogyra, by combining in vitro and in vivo approaches to unveil the nature of its barite microcrystals. Scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDXS) observations on simply dried samples revealed that the number and size of barite crystals were related to the barium concentration in the media. Additionally, their morphology showed a crystallographic face (011), which is not normally observed, suggesting the influence of organic molecules on the growth kinetics. The critical point drying method was used to preserve the internal and external structures of Spirogyra cells for SEM imaging. Crystals were found adjacent to the cytoplasmic membrane, near chloroplasts and fibrillary network. In vivo optical microscopy and Raman tweezer microspectroscopy in living cells showed that barite microcrystals are optically visible and follow cytoplasmic streaming. These results led us to propose that barite formation in Spirogyra occurs in the cytoplasm where barium and sulphate are both available: barium supplied non-selectively through the active transport of the divalent cations needed for actin polymerisation, and sulphate because necessary for amino acid biosynthesis in chloroplasts.

Plasmonic Cavities and Individual Quantum Emitters in the Strong Coupling Limit

ConspectusThe interaction of emitters with plasmonic cavities (PCs) has been studied extensively during the past decade. Much of the experimental work has focused on the weak coupling regime, manifested most importantly by the celebrated Purcell effect, which involves a modulation of the spontaneous emission rate of the emitter due to interaction with the local electromagnetic density of states. Recently, there has been a growing interest in studying hybrid emitter-PC systems in the strong-coupling (SC) regime, in which the excited state of an emitter hybridizes with that of the PC to generate new states termed polaritons. This phenomenon is termed vacuum Rabi splitting (VRS) and is manifested in the spectrum through splitting into two bands.In this Account, we discuss SC with PCs and focus particularly on work from our lab on the SC of quantum dots (QDs) and plasmonic silver bowtie cavities. As bowtie structures demonstrate strong electric field enhancement in their gaps, they facilitate approaching the SC regime and even reaching it with just one to a few emitters placed there. QDs are particularly advantageous for such studies, due to their significant brightness and long lifetime under illumination. VRS was observed in our lab by optical dark-field microspectroscopy even in the limit of individual QDs. We further used electron energy loss spectroscopy, a near-field spectroscopic technique, to facilitate measuring SC not only in bright modes but also in subradiant, dark plasmonic modes. Dark modes are expected to live longer than bright modes and therefore should be able to store electromagnetic energy for longer times.Photoluminescence (PL) is another useful observable for probing the SC regime at the single-emitter limit, as shown by several laboratories. We recently used Hanbury Brown and Twiss interferometry to demonstrate the quantum nature of PL from QDs within PCs, verifying that the measurements are indeed from one to three QDs. Further spectroscopic studies of QD-PC systems in fact manifested several surprising features, indicating discrepancies between scattering and PL spectra. These observations pointed to the contribution of multiple excited states. Indeed, using model simulations based on an extended Jaynes–Cummings Hamiltonian, it was found that the involvement of a dark state of the QDs can explain the experimental findings. Given that bright and dark states couple to the cavity with different degrees of coupling strength, the PC affects in a different manner each excitonic state. This yields complex relaxation pathways and interesting dynamics.Future work should allow us to increase the QD-PC coupling deeper into the SC regime. This will pave the way to exciting applications including the generation of single-photon sources and studies of cavity-induced coherent interactions between emitters.

Femtosecond laser nanoprinting of anisotropic plasmonic surfaces: coloration and anticounterfeiting.

An anisotropic plasmonic surface of nanoellipsoid arrays is successfully fabricated on an Au film using slit-shaping-based femtosecond laser nanoprinting. The size and orientation of the nanoellipsoid can be exquisitely and flexibly controlled by adjusting the width and direction of the slit and the laser pulse energy. By dark-field optical micro-spectroscopy, anisotropic plasmonic color rendering as well as resonant light scattering from the lateral and vertical modes are experimentally and theoretically investigated in the visible spectral range. In addition, prospective use in the fields of steganographic encryption and multidimensional optical multiplexing is also proposed.

Co-localized line-field confocal optical coherence tomography and confocal Raman microspectroscopy for three-dimensional high-resolution morphological and molecular characterization of skin tissues ex vivo.

Line-field confocal optical coherence tomography (LC-OCT) is an optical modality that provides three-dimensional (3D) images of the skin at cellular resolution. Confocal Raman microspectroscopy (CRM) is a label-free optical technique that can provide point measurement of the molecular content of the skin. This work presents a method to co-localize LC-OCT and CRM acquisitions for morpho-molecular analysis of ex vivo skin tissues at cellular level. The co-localization method allows acquisition of Raman spectra at specific locations in a sample identified from a 3D LC-OCT image, with an accuracy of ± 20 µm. The method was applied to the characterization of tattooed skin biopsies with adverse tattoo reactions. LC-OCT images allowed to target specific regions in the biopsies where the presence of tattoo ink was revealed by detection of the Raman signature of ink pigments. Micrometer-sized foreign bodies of various materials as well as inflammatory cells were also identified within the biopsies. From these results, we demonstrate the value of the LC-OCT-CRM co-localization method and its potential for future ex vivo analysis of suspicious skin lesions.

Raman identification of the different glazing technologies of Blue-and-White Ming porcelains

Shards of Blue-and-White Ming porcelain from shipwrecks of Portuguese ships found on the coasts of South Africa plus a shard from Mombasa (Kenya) were analyzed by optical microscopy, SEM-EDS and Raman microspectroscopy (458 nm). Whereas the Raman signature of porcelain body paste compositions which are based on mullite, quartz and an amorphous phase with the minor presence of anatase and feldspar are very comparable whatever the variable alumina content, at least two types of glazes are observed: a high-temperature soda-potassium glaze, and glazes richer in calcium and similar to a celadon glaze (with the possibility of wollastonite formation). The blue decoration is obtained with a material rich in manganese typical of the Ming productions. Some shards exhibit a two-layer glaze and the blue decoration is either placed under-glaze, or in-glaze as found in the Vietnamese productions of the same period. Previous assignments of the Raman signature of feldspar to cobalt aluminate are now not favoured (blue colour is obtained with Co2+ ions dissolved in the glassy silicate) and several black spots exhibit the characteristic spectrum of an epsilon Fe2O3 phase being present.

Optical coherence tomography-guided confocal Raman microspectroscopy for rapid measurements in tissues.

We report a joint system with both confocal Raman spectroscopy (CRS) and optical coherence tomography (OCT) modules capable of quickly addressing the region of interest in a tissue for targeted Raman measurements from OCT. By using an electrically tunable lens in the Raman module, the focus of the module can be adjusted to address any specific depth indicated in an OCT image in a few milliseconds. We demonstrate the performance of the joint system in the depth dependent measurements of an ex vivo swine tissue and in vivo human skin. This system can be useful in measuring samples embedded with small targets, for example, to identify tumors in skin in vivo and assessment of tumor margins, in which OCT can be used to perform initial real-time screening with high throughput based on morphological features to identify suspicious targets then CRS is guided to address the targets in real time and fully characterize their biochemical fingerprints for confirmation.

The Technology Transfer from Europe to China in the 17th-18th Centuries: Non-Invasive On-Site XRF and Raman Analyses of Chinese Qing Dynasty Enameled Masterpieces Made Using European Ingredients/Recipes.

Two masterpieces of the Qing Dynasty (1644–1912 CE), one in gilded brass (incense burner) decorated with cloisonné enamels stylistically attributed to the end of the Kangxi Emperor’s reign, the other in gold (ewer offered by Napoleon III to the Empress as a birthday present), decorated with both cloisonné and painted enamels bearing the mark of the Qianlong Emperor, were non-invasively studied by optical microscopy, Raman microspectroscopy and X-ray microfluorescence spectroscopy (point measurements and mapping) implemented on-site with mobile instruments. The elemental compositions of the metal substrates and enamels are compared. XRF point measurements and mappings support the identification of the coloring phases and elements obtained by Raman microspectroscopy. Attention was paid to the white (opacifier), blue, yellow, green, and red areas. The demonstration of arsenic-based phases (e.g., lead arsenate apatite) in the blue areas of the ewer, free of manganese, proves the use of cobalt imported from Europe. The high level of potassium confirms the use of smalt as the cobalt source. On the other hand, the significant manganese level indicates the use of Asian cobalt ores for the enamels of the incense burner. The very limited use of the lead pyrochlore pigment (European Naples yellow recipes) in the yellow and soft green cloisonné enamels of the Kangxi incense burner, as well as the use of traditional Chinese recipes for other colors (white, turquoise, dark green, red), reinforces the pioneering character of this object in technical terms at the 17th–18th century turn. The low level of lead in the cloisonné enamels of the incense burner may also be related to the use of European recipes. On the contrary, the Qianlong ewer displays all the enameling techniques imported from Europe to obtain a painted decoration of exceptional quality with the use of complex lead pyrochlore pigments, with or without addition of zinc, as well as cassiterite opacifier.

Insights into the porous structure of surfactant-promoted gas hydrate

We show how insights into the porous structure of surfactant-promoted gas hydrate can be obtained from two types of experiments that probe very different length scales. On the one hand, the observation of an existing porous hydrate being imbibed with the aqueous surfactant solution gives information as to the porosity – inferred from the volume increase – and the average pore size – inferred from the imbibition rate. On the other hand, direct pore visualization and determination of the fluid and hydrate contents at the micron scale are obtained by optical microscopy and Raman microspectroscopy. We observe a porosity in the range of 60–70% and pore sizes of about 20–30 μm for a porous methane hydrate formed from a 500 ppmw SDS (sodium dodecyl sulfate) solution under moderate subcooling.

Morpho-Molecular Metabolic Analysis and Classification of Human Pituitary Gland and Adenoma Biopsies Based on Multimodal Optical Imaging.

Simple SummaryThe pituitary gland governs the function of nearly all endocrine glands and pituitary oncogenesis often distorts the hormonal balance. For optimal surgical cure it is crucial to discriminate pathological tissue from intact pituitary gland. Our multimodal imaging approach allows for morpho-molecular metabolic analysis and discrimination of pituitary gland and adenomas combining different complementary techniques such as optical coherence tomography (OCT), multiphoton microscopy (MPM) and line scan Raman microspectroscopy (LSRM). Radiomics as well as analysis of spectroscopic features allows enhanced discrimination of pituitary gland and adenomas and, furthermore, classification of pituitary adenoma subtypes.Pituitary adenomas count among the most common intracranial tumors. During pituitary oncogenesis structural, textural, metabolic and molecular changes occur which can be revealed with our integrated ultrahigh-resolution multimodal imaging approach including optical coherence tomography (OCT), multiphoton microscopy (MPM) and line scan Raman microspectroscopy (LSRM) on an unprecedented cellular level in a label-free manner. We investigated 5 pituitary gland and 25 adenoma biopsies, including lactotroph, null cell, gonadotroph, somatotroph and mammosomatotroph as well as corticotroph. First-level binary classification for discrimination of pituitary gland and adenomas was performed by feature extraction via radiomic analysis on OCT and MPM images and achieved an accuracy of 88%. Second-level multi-class classification was performed based on molecular analysis of the specimen via LSRM to discriminate pituitary adenomas subtypes with accuracies of up to 99%. Chemical compounds such as lipids, proteins, collagen, DNA and carotenoids and their relation could be identified as relevant biomarkers, and their spatial distribution visualized to provide deeper insight into the chemical properties of pituitary adenomas. Thereby, the aim of the current work was to assess a unique label-free and non-invasive multimodal optical imaging platform for pituitary tissue imaging and to perform a multiparametric morpho-molecular metabolic analysis and classification.

Revealing Inflammatory Indications Induced by Titanium Alloy Wear Debris in Periprosthetic Tissue by Label-Free Correlative High-Resolution Ion, Electron and Optical Microspectroscopy.

The metallic-associated adverse local tissue reactions (ALTR) and events accompanying worn-broken implant materials are still poorly understood on the subcellular and molecular level. Current immunohistochemical techniques lack spatial resolution and chemical sensitivity to investigate causal relations between material and biological response on submicron and even nanoscale. In our study, new insights of titanium alloy debris-tissue interaction were revealed by the implementation of label-free high-resolution correlative microscopy approaches. We have successfully characterized its chemical and biological impact on the periprosthetic tissue obtained at revision surgery of a fractured titanium-alloy modular neck of a patient with hip osteoarthritis. We applied a combination of photon, electron and ion beam micro-spectroscopy techniques, including hybrid optical fluorescence and reflectance micro-spectroscopy, scanning electron microscopy (SEM), Energy-dispersive X-ray Spectroscopy (EDS), helium ion microscopy (HIM) and micro-particle-induced X-ray emission (micro-PIXE). Micron-sized wear debris were found as the main cause of the tissue oxidative stress exhibited through lipopigments accumulation in the nearby lysosome. This may explain the indications of chronic inflammation from prior histologic examination. Furthermore, insights on extensive fretting and corrosion of the debris on nm scale and a quantitative measure of significant Al and V release into the tissue together with hydroxyapatite-like layer formation particularly bound to the regions with the highest Al content were revealed. The functional and structural information obtained at molecular and subcellular level contributes to a better understanding of the macroscopic inflammatory processes observed in the tissue level. The established label-free correlative microscopy approach can efficiently be adopted to study any other clinical cases related to ALTR.