Casein micelles (CM) are colloidal phospho-protein-mineral complexes naturally present in milk. Cooling inducesβ-casein dissociation from CM. The aim of the present research is to improve curcumin encapsulation in β-casein depleted CM. This study proposes an extraction and separation process including cooling, low acidification (pH 5.8) and membrane filtration at a pilot scale allowing a removal of 45 ±5 % of β-casein fraction from CM.Special care was taken to preserve the CM integrity. Investigation of the depleted CM topography by atomic force microscopy (AFM) reveals a swelling of the micellar structure with a mean width of 193 ±10 nm and a mean height of 80 ±5 nm. Concurrently, exploration of the elastic properties displays a stiffer nanomechanicalsignature compared to native CM, with a mean elasticity modulus of 195 ±17 kPa. The β-caseins extracted from CM were bound to curcumin prior to their encapsulation in depleted CM. This new encapsulation strategy was compared to two other methods, and the results show that it significantly increases the binding efficiency of curcumin to CM.

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 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 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 the 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 crosslinking agents further modulate collagen fibril formation and its mechanical properties.

The high chemical specificity, minimal sample preparationand the ability to use advanced optical technologies in thevisible or near-infrared spectral range have led in the lastyears to an increase in the biomedical applications of Ramanspectroscopy. Raman spectroscopy measures the inelasticscattering of light by vibrating molecules, providing thechemical fingerprints of cells, tissues or biofluids. However,as Raman scattering efficiency is quite low, requiringrelatively long acquisition times, new technologies based onBroadband Coherent Anti-Stokes Raman scattering(BCARS) was proposed [1]. This coherent Raman imagingtechnique is based on the detection of the fingerprint regionof molecules (500-1800 cm-1), as well as the higher-energyCH-/OH-stretch region (~2,700 cm−1 to 3,300 cm−1) andcould probe multiple Raman transitions simultaneously toallow imaging of biological tissues with improved molecularcontrast. When compared to spontaneous Ramanmicroscopy, BCARS microscopy provided 10-100x fasterimage acquisition for quantitative and qualitative assessmentof pharmaceuticals at much higher spatiochemical resolutionand with spectra of much higher signal-to noise ratio [2]. Inthis talk, I will present the principles of the BCARSmicroscopy we’ve developed in our laboratory and someexamples of biological applications. BCARS imaging is usedto evaluate changes underlying the lesion of a spinal cordinjury in a murine model. After injury the axonalregeneration in spinal cord is inhibited by a glial and afibrous scar, acting as a physical and chemical barrier.Recently we have reported that the signal exhibited byfibrillar collagen enabled to specifically monitor it as abiomarker of a spinal cord lesion [3]. However, the injuryand the scarring process involves also other elements as themyelin sheets, microglia, astrocytes and extracellular matrixcomponents. We demonstrate the versatility of BCARSimaging to assess the additional structural and metabolicmodifications of the injured tissue in excised murine spinalcord samples at different time points (1, 2, 4, 6 weeks) afterinjury.1) Camp Jr CH et al. Nat. Photonics 8(8), 627 (2014).2) Hartshorn CM et al. Anal. Chem. 85(17), 8102 (2013)3) C. Manesco, et al. Nanomedicine: Nanotechnology,Biology, and Medicine 53 (2023) 102699.

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 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 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 the 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.

ABSTRACT 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.

Broadband CARS imaging is used to evaluate changes underlying the lesion of a spinal cord injury in a murine model. After injury the axonal regeneration in spinal cord is partly inhibited by the glial scar, acting as a physical and chemical barrier. Recently we have reported that the SHG signal exhibited by fibrillar collagen enabled to specifically monitor it as a biomarker of a spinal cord lesion1. However, the injury and the scarring process involves also other elements as the myelin sheets, microglia, astrocytes and extracellular matrix components. We assess the additional structural and metabolic modifications of the injured tissue via BCARS imaging of excised murine spinal cord tissue at different time points (72h, 1, 2, 6 weeks) after injury. BCARS is based on the detection of the fingerprint region of molecules (500-1500 cm-1) and could probe multiple Raman transitions simultaneously, to allow a “chemical” imaging of biological tissues with improved molecular contrast2. When compared to spontaneous Raman microscopy, BCARS microscopy provided 10-100x faster image acquisition for quantitative and qualitative assessment of pharmaceuticals at much higher spatio-chemical resolution and with spectra of much higher signal-to noise ratio.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects both upper and lower motor neurons. As motor neurons degenerate, they lose their connection to muscle cells, leading to the onset of fasciculations and muscle atrophy. Eventually, ALS results in paralysis, with symptoms progressively worsening over time. The disease typically leads to death within 3–5 years after diagnosis. Currently, there is no definitive cure. Understanding the morphological and mechanical modifications in skeletal muscle fibers of an ALS mouse model (SOD1-G93A) could be crucial for uncovering the molecular mechanisms underlying the disease.In this study, second harmonic generation (SHG) multiphoton microscopy and atomic force microscopy (AFM) were used to evaluate sarcomere periodicity, stiffness, and orientation in gastrocnemius muscle fibers from symptomatic SOD1-G93A mice (90 days), comparing them to wild-type (WT) controls. Our results reveal a significant reduction in sarcomere periodicity in SOD1-G93A mice compared to WT controls. Furthermore, AFM data indicate the presence of two populations of muscle fibers: slower Type-I and faster Type-II fibers. Notably, SOD1-G93A tissue exhibits a dramatic softening of fast fibers, while not statistical difference was detected for slow fibers. This result could indicate that ALS, at this stage of the disease, selectively affects skeletal muscle fibers specialized for fast contraction.These alterations in mechanical properties and morphology reflect structural and molecular changes in the sarcomeres, suggesting a change in inner structure of the main structural proteins.