Collagen, the primary structural protein in the extracellular matrix, plays a critical role in tissue architecture and mechanical integrity. This study investigates the structural and nonlinear optical properties of atelocollagen (Acol) and telocollagen (Tcol) in response to pH variations by using second harmonic scattering (SHS), polarization-resolved SHS (P-SHS), and atomic force microscopy (AFM). AFM imaging revealed distinct morphological differences, with Acol forming wavy, non-cross-linked, and randomly arranged fibers, while Tcol exhibited a more interconnected, mesh-like fibrillar network. SHS and P-SHS measurements further confirmed these differences, indicating significant variations in hyperpolarizability and depolarization ratio (DR) between Acol and Tcol. The results suggest that telopeptides play a crucial role in collagen fibrillogenesis, influencing the molecular symmetry and optical responses. The observed differences in nonlinear optical properties highlight the potential of SHS as a powerful tool for characterizing collagen self-assembly mechanisms. These findings contribute to a broader understanding of collagen organization in biological and biomaterial contexts, with implications for tissue engineering, fibrosis research, and regenerative medicine. Future work should explore how external factors, such as ionic strength and cross-linking agents, further modulate collagen fibril formation and its mechanical properties.

Collagen is a key protein in mammals that maintains structural integrity within tissues. A failure in fibrillar collagen reorganization can induce cancer or fibrosis formation, such as in spinal cord injury (SCI), where the healing process after the initial trauma leads to the formation of scar tissue, which includes fibrosis. As there is no current treatment targeting the fibrotic process directly, a better understanding of collagen properties can thus help to apprehend malignant states. Characterization of collagen fibers has been widely explored on second-harmonic generation (SHG) images, due to the label-free nature of the SHG imaging technique. It has been performed with various fibers extraction methods such as curvelet transform (CT) implemented in the open-source software CurveAlign. However, when it comes to investigating undergrowth collagen fibers (collagen fibers that are still under reorganization) as observed in SCI, the CT method becomes complex to tune for nonadvanced users in order to properly segment the fibers. To improve collagen detection in the case of undergrowth fibers, we propose a methodology based on the fingerprint enhancement (FP-E) algorithm that requires fewer user input parameters and is less time-consuming. Our method was extensively tested on SHG data from injured spinal cord samples. We obtained metrics that depicted changes in collagen organization over time, particularly a significant increase in fiber density, demonstrating the FP-E algorithm was properly adapted to address the evolution of collagen properties after SCI. Besides the simpler tuning of the method compared to commonly used software, the combination with further characterization of the extracted fibers could lead to consider fibrillar collagen as a biomarker in diseases where fibers are under development. The FP-E algorithm is provided in the article.

The rheological properties of the periodontal ligament are key parameters to understand the homeostatic stability of the tooth supporting apparatus. The objective of this study is to lay new insights on the rheological properties and structural information of different regions in murine periodontal ligament by using atomic force (AFM) and multi-photon microscopy (MPM). A significant variation in elasticity of different regions was measured. The elasticity of periodontal ligament showed a significant tendency to become softer towards the furcation, the nearest area to the center of resistance of the tooth. This can open a new prospective for connecting the rheological adaptation of periodontal ligament to the tooth geometry that defines the center of resistance. Another important finding revealed by the second harmonic generation (SHG) signal exhibited by collagen fibers and measured by MPM is that the orientation of fibers in the furcation region can both provide space for tooth vertical movement with high compressive loads and prevent horizontal tooth movement. Additionally, it was found that the dispersion of the angles at different levels of cutting indicates homogeneity in the directionality of fibers across different regions. These results provide an accurate description of the rheological properties and structural information of periodontal ligament, which can serve as a base for comparison with other local and systemic diseases that may influence the periodontal ligament.

Spinal cord injury is a dramatic disease leading to severe motor, sensitive and autonomic impairments. After injury the axonal regeneration is partly inhibited by the glial scar, acting as a physical and chemical barrier. The scarring process involves microglia, astrocytes and extracellular matrix components, such as collagen, constructing the fibrotic component of the scar. To investigate the role of collagen, we used a multimodal label-free imaging approach combining multiphoton and atomic force microscopy. The second harmonic generation signal exhibited by fibrillar collagen enabled to specifically monitor it as a biomarker of the lesion. An increase in collagen density and the formation of more tortuous fibers over time after injury are observed. Nano-mechanical investigations revealed a noticeable hardening of the injured area, correlated with collagen fibers' formation. These observations indicate the concomitance of important structural and mechanical modifications during the fibrotic scar evolution.

Sarcomere length (SL) and its variation along the myofibril strongly regulate integrated coordinated myocyte contraction. It is therefore important to obtain individual SL properties. Optical imaging by confocal fluorescence (for example, using ANEPPS) or transmitted light microscopy is often used for this purpose. However, this allows for the visualization of structures related to Z-disks only. In contrast, second-harmonic generation (SHG) microscopy visualizes A-band sarcomeric structures directly. Here, we compared averaged SL and its variability in isolated relaxed rat cardiomyocytes by imaging with ANEPPS and SHG. We found that SL variability, evaluated by several absolute and relative measures, is two times smaller using SHG vs. ANEPPS, while both optical methods give the same average (median) SL. We conclude that optical methods with similar optical spatial resolution provide valid estimations of average SL, but the use of SHG microscopy for visualization of sarcomeric A-bands may be the "gold standard" for evaluation of SL variability due to the absence of optical interference between the sarcomere center and non-sarcomeric structures. This contrasts with sarcomere edges where t-tubules may not consistently colocalize to Z-disks. The use of SHG microscopy instead of fluorescent imaging can be a prospective tool to map sarcomere variability both in vitro and in vivo conditions and to reveal its role in the functional behavior of living myocardium.

Spinal cord injuries (SCI) affect between 2.5 and 4 million patients worldwide, with no current curative treatment. To understand the mechanisms underlying the absence of spontaneous regeneration following injury, we are combining the non-linear multiphoton microscopy (MPM) technique with force measurements via atomic force microscopy (AFM), in a mouse model, to monitor the glial scar, a scar that inhibits the axonal regeneration by forming a physical and chemical barrier composed mainly of astrocytes and microglia. We recorded 2-photon excited fluorescence (2PEF) and second harmonic generation (SHG) signals of excised mice SC injured tissues in MPM at 72h, 1week and 6 weeks post-lesion, and further performed polarization dependent measurements of the SHG signal to assess the preferential orientation of the collagen bundles. Our MPM images revealed a strong SHG signal at 1 week post injury, due to the formation of fibrillary collagen fibers (collagen type I) by the injury site. The SHG signal was increased at 6 weeks after injury, and associated with (1) a higher fiber density (2) a shorter fiber length and less fibers oriented in the same direction. AFM based force spectroscopy measurements, performed at the same post-lesion time-points to map the elastic properties of the spared grey and white matters and injured (lesion) parts of the tissue, suggested an increase of the lesion area stiffness over time. These results together indicate the presence of a fibrotic process seven days after injury, that is further increased at later time points. We similarly started to investigate the effect of a treatment (pharmacological transient depletion of microglia/macrophage proliferation) in mice that underwent SCI. Our preliminary results suggested an increase in fibers length in treated tissues, as well as a reduction of the collagen extension around the injury site.

The molecular and cellular mechanisms associated with tissue degradation or regeneration in an infectious context are poorly defined. Herein, we explored the role of macrophages in orchestrating either tissue regeneration or degradation in zebrafish embryos pre-infected with the fish pathogen Mycobacterium marinum. Zebrafish were inoculated with different infectious doses of M. marinum prior to fin resection. While mild infection accelerated fin regeneration, moderate or severe infection delayed this process by reducing blastemal cell proliferation and impeding tissue morphogenesis. This was correlated with impaired macrophage recruitment at the wound of the larvae receiving high infectious doses. Macrophage activation characterized, in part, by a high expression level of tnfa was exacerbated in severely infected fish during the early phase of the regeneration process, leading to macrophage necrosis and their complete absence in the later phase. Our results demonstrate how a mycobacterial infection influences the macrophage response and tissue regenerative processes.

Duchenne muscular dystrophy (DMD) is a debilitating disorder related to dystrophin encoding gene mutations, often associated with dilated cardiomyopathy. However, it is still unclear how dystrophin deficiency affects cardiac sarcomere remodeling and contractile dysfunction. We employed second harmonic generation (SHG) microscopy, a nonlinear optical imaging technique that allows studying contractile apparatus organization without histologic fixation and immunostaining. Images were acquired on alive DMD (mdx) and wild type cardiomyocytes at different ages and at various external calcium concentrations. An automated image processing was developed to identify individual myofibrils and extract data about their organization. We observed a structural aging-dependent remodeling in mdx cardiomyocytes affecting sarcomere sinuosity, orientation and length that could not be anticipated from standard optical imaging. These results revealed for the first time the interest of SHG to evaluate the intracellular and sarcomeric remodeling of DMD cardiac tissue in an age-dependent manner that could participate in progressive contractile dysfunction.

The ZnSe-ZnTe combination is a potential candidate for the realisation of visible light-emitting devices. The lattice mismatch between bulk ZnSe and bulk ZnTe is important (~ 8%). Therefore, their hetero-structures are strained and high quality superlattices will only be grown if having small periods. This prescription can be fulfilled in the case of metal organic vapour phase epitaxy (MOVPE) growth by combining triethylamine dimethyl zinc adduct with diisopropyl telluride as precursors for the growth of the ZnTe layers. The growth of high quality ZnTe can then be performed at a temperature of 300°C, close to the best MOVPE-growth temperature for ZnSe (280°C). Lowering the growth temperature of ZnTe to this value, we could thus obtain sharp interfaces. This work reports on ZnSe-ZnTe superlattices grown on ZnSe and ZnTe buffers deposited on GaAs substrates. We demonstrate that the stokes-shift between the reflectance and photoluminescence features (~ 40 meV) measured when the thickness of ZnSe layers does not exceed 20 Å, drastically increases for layer thicknesses beyond this critical value. This, we interpret in terms of the onset of plastic relaxation which favours tellurium diffusion in the ZnSe slices. Then photoluminescence spectra broaden (contributions of trapped-excitons dominate), and observation of free excitons in reflectance become impossible. We have studied in detail the optical properties of the superlattices and compared our findings with the predictions of a multiband envelope function calculation. We show that both zone centre excitons as well as excitons associated with the miniband dispersions (saddle-point excitons) are observed in these superlattices.

In recent years, fluorescent nanodiamond (fND) particles containing nitrogen-vacancy (NV) centers gained recognition as an attractive probe for nanoscale cellular imaging and quantum sensing. For these applications, precise localization of fNDs inside of a living cell is essential. Here we propose such a method by simultaneous detection of the signal from the NV centers and the spectroscopic Raman signal from the cells to visualize the nucleus of living cells. However, we show that the commonly used Raman cell signal from the fingerprint region is not suitable for organelle imaging in this case. Therefore, we develop a method for nucleus visualization exploiting the region-specific shape of C-H stretching mode and further use k-means cluster analysis to chemically distinguish the vicinity of fNDs. Our technique enables, within a single scan, to detect fNDs, distinguish by chemical localization whether they have been internalized into cell and simultaneously visualize cell nucleus without any labeling or cell-fixation. We show for the first time spectral colocalization of unmodified high-pressure high-temperature fND probes with the cell nucleus. Our methodology can be, in principle, extended to any red- and near-infrared-luminescent cell-probes and is fully compatible with quantum sensing measurements in living cells.

The development of new diagnostic technologies based on the light scattering and autofluorescence properties of dental tissues is required to improve the diagnostic ability of initial caries lesions earlier than previously done and promoting the potential of treatment without surgical intervention. The aim of this study is to correlate fluorescence-based results provided by multiphoton microscopy (MPM) with confocal Raman microscopy records using phosphate level at 960 cm–1 and the organic matrix at ∼2,931 cm–1 in healthy and demineralized human enamel. Measurements on 14 teeth were made using two incident lights of different wavelengths, released by confocal Raman microscopy and MPM. Raman phosphate peak intensity at 960 cm–1 along with organic to mineral ratio at (2,931/430 cm–1) and nonlinear optical signals (second harmonic generation [SHG] and intrinsic two-photon excited fluorescence [I2PEF]) were recorded from the demineralized and healthy enamel sites. Raman spectral maps showed that the higher the organic/mineral ratio in the demineralized enamel, the lower the intensity of mineral component in the same zone. MPM revealed new optical indicators of carious lesion as shown by the presence of a red-shifted fluorescence peak in the 650- to 750-nm area of the fluorescence spectrum of demineralized enamel. Moreover, on sample regions with insignificant autofluorescence, the emergence of the SHG signal could be noted. By comparing I2PEF images with the structural motifs observed by the confocal Raman imaging system, the morphological similarity of the acquired images was quite evident. Any change in the I2PEF spectra reflects alterations in the chemical composition of enamel. These findings may provide an important basis for potentially valuable applications of photonic tools in the clinical diagnosis of tooth pathological conditions, besides exposing the fundamental role of organic matrix in enamel integrity and reparation.

Chronic kidney disease is characterized by a gradual decline in renal function that progresses toward end-stage renal disease. Podocytes are highly specialized glomerular epithelial cells which form with the glomerular basement membrane (GBM) and capillary endothelium the glomerular filtration barrier. GBM is an extracellular matrix (ECM) that acts as a mechanical support and provides biophysical signals that control normal podocytes behavior in the process of glomerular filtration. Thus, the ECM stiffness represents an essential characteristic that controls podocyte function. Hydrolyzed Polyacrylamide (PAAm) hydrogels are smart polyelectrolyte materials. Their biophysical properties can be tuned as desired to mimic the natural ECM. Therefore, these hydrogels are investigated as new ECM-like constructs to engineer a podocyte-like basement membrane that forms with cultured human podocytes a functional glomerular-like filtration barrier. Such ECM-like PAAm hydrogel construct will provide unique opportunity to reveal podocyte cell biological responses in an in vivo-like setting by controlling the physical properties of the PAAm membranes. In this work, Hydrolyzed PAAm scaffolds having different stiffness ranging between 0.6-44 kPa are prepared. The correlation between the hydrogel structural and mechanical properties and Podocyte morphology, elasticity, cytoskeleton reorganization, and podocin expression is evaluated. Results show that hydrolyzed PAAm hydrogels promote good cell adhesion and growth and are suitable materials for the development of future 3D smart scaffolds. In addition, the hydrogel properties can be easily modulated over a wide physiological range by controlling the cross-linker concentration. Finally, tuning the hydrogel properties is an effective strategy to control the cells function. This work addressed the complexity of podocytes behavior which will further enhance our knowledge to develop a kidney-on-chip model much needed in kidney function studies in both healthy and diseased states.

Incipient enamel lesions represent an early phase of dental caries. They can be produced in-vitro by pH cycling Raman spectral maps showed that the higher the organic/mineral ratio in the demineralized enamel, the lower is the intensity of mineral content in the same zone. Moreover, the evident red shift of the two-photon excited fluorescence (2PEF) spectrum from carious enamel, compared to that from sound enamel is typical of carious lesion formation. Second harmonic generation (SHG) signal was also observed in the lesion zone. The obtained results demonstrate a great potential for Raman, 2PEF and SHG photonic signals in diagnostic of incipient carious lesions.

Extensive use of porous silicon (PSi) for tissue engineering is due to its convenient properties as it is both nontoxic and bioresorbable. Moreover, PSi surface modification is an important step to enhance cell adhesion and proliferation. In this work, a combination of optical and electrochemical studies is performed to elaborate a suitable PSi multilayer substrate for cell culture. For this study, we modified PSi surface by silanization and antibody grafting (APTES-anti STRO1), the 12-mer specific peptide to silicon p + type coating and the peptide modified with the antibody recognition sequence. Electrochemical characterization of PSi multilayers is performed to investigate its electrical behavior, determine the optimal measuring conditions and reveal the most stable PSi surfaces. Then, the behavior of dental pulp stem cells (DPSC) was investigated on various modified PSi surfaces. An electrochemical method was applied for the first time monitoring the electrical behavior of stem cell adhesion. The cells electrochemical behavior depends on the nature of the surface coating and the peptide-anti STRO1 improved adhesion and cell spreading onto the PSi surface compared to bare surface and the one coated with the peptide. Fluorescent microscopy revealed that all surface modification methods enhance cell adhesion compared to the bare PSi surface. An increased cell number is observed on APTES-anti STRO1, peptide and peptide-anti STRO1 coated PSi. The peptide-anti STRO1 provided the best cell proliferation results suggesting the improved accessibility of the recognition fragment of the antibody anti-STRO1.