Multiphoton Label-Free ex-vivo imaging using a custom-built dual-wavelength microscope with chromatic aberrations compensation

Label-Free Multiphoton Microscopy is a very powerful optical microscopy that can be applied to study samples with no need for exogenous fluorescent probes, keeping the main benefits of a Multiphoton approach, like longer penetration depths and intrinsic optical sectioning, while opening the possibility of serial examinations with different kinds of techniques. Among the many variations of Label-Free MPM, Higher Harmonic Generation (HHG) is one of the most intriguing due to its generally low photo-toxicity, which enables the examination of specimens particularly susceptible to photo-damages. HHG and common Two-Photon Microscopy (TPM) are well-established techniques, routinely used in several research fields. However, they require a significant amount of fine-tuning in order to be fully exploited and, usually, the optimized conditions greatly differ, making them quite difficult to perform in parallel without any compromise on the extractable information. Here we present our custom-built Multiphoton microscope capable of performing simultaneously TPM and HHG without any kind of compromise on the results thanks to two, separate, individually optimized laser sources with full chromatic aberration compensation. We also apply our setup to the examination of a plethora of ex vivo samples in order to prove the significant advantages of our approach

Advanced microscopy analysis of the micro-nanoscale architecture of human menisci

The complex inhomogeneous architecture of the human meniscal tissue at the micro and nano scale in the absence of artefacts introduced by sample treatments has not yet been fully revealed. The knowledge of the internal structure organization is essential to understand the mechanical functionality of the meniscus and its relationship with the tissue’s complex structure. In this work, we investigated human meniscal tissue structure using up-to-date non-invasive imaging techniques, based on multiphoton fluorescence and quantitative second harmonic generation microscopy complemented with Environmental Scanning Electron Microscopy measurements. Observations on 50 meniscal samples extracted from 6 human menisci (3 lateral and 3 medial) revealed fundamental features of structural morphology and allowed us to quantitatively describe the 3D organisation of elastin and collagen fibres bundles. 3D regular waves of collagen bundles are arranged in “honeycomb-like” cells that are comprised of pores surrounded by the collagen and elastin network at the micro-scale. This type of arrangement propagates from macro to the nanoscale

The three dimensional microstructural network of elastin, collagen and cells in Achilles tendons

Similar to most biological tissues, the biomechanical and functional characteristics of the Achilles tendon are closely related to its composition and microstructure. It is commonly reported that type I collagen is the predominant component of tendons and is mainly responsible for the tissue's function. Although elastin has been found in varying proportions in other connective tissues, previous studies report that tendons contain very small quantities of elastin. However, the morphology of and the microstructural relationship among the elastic fibres, collagen and cells in tendon tissue have not been well examined. We hypothesize the elastic fibres, as another fibrillar component in the extracellular matrix, have a unique role in mechanical functions and microstructural arrangement in Achilles tendons. Using confocal and Second Harmonic Generation (SHG) imaging techniques, this study examined the 3-dimensional microstructure of the collagen, elastin and cells in the mid-portion of hydrated rabbit Achilles tendons. It has been shown that elastic fibres present a close connection with the tenocytes. The close relationship of the three components has been revealed as a distinct, integrated and complex microstructural network. Notably, a "spiral" structure within fibril bundles in Achilles tendons was observed in some samples in specialized regions. This study substantiates the hierarchical system of the spatial microstructure of tendon, including the mapping of collagen, elastin and tenocytes, with 3-dimensional confocal images

The micromechanics of the superficial zone of articular cartilage.

Journal ArticleOBJECTIVE: To investigate the relationships between the unique mechanical and structural properties of the superficial zone of articular cartilage on the microscopic scale. DESIGN: Fresh unstained equine metacarpophalangeal cartilage samples were mounted on tensile and compressive loading rigs on the stage of a multiphoton microscope. Sequential image stacks were acquired under incremental loads together with simultaneous measurements of the applied stress and strain. Second harmonic generation was used to visualise the collagen fibre network, while two photon fluorescence was used to visualise elastin fibres and cells. The changes visualised by each modality were tracked between successive loads. RESULTS: The deformation of the cartilage matrix was heterogeneous on the microscopic length scale. This was evident from local strain maps, which showed shearing between different regions of collagen under tensile strain, corrugations in the articular surface at higher tensile strains and a non-uniform distribution of compressive strain in the axial direction. Chondrocytes elongated and rotated under tensile strain and were compressed in the axial direction under compressive load. The magnitude of deformation varied between cells, indicating differences in either load transmission through the matrix or the mechanical properties of individual cells. Under tensile loading the reorganisation of the elastin network differed from a homogeneous elastic response, indicating that it forms a functional structure. CONCLUSIONS: This study highlights the complexity of superficial zone mechanics and demonstrates that the response of the collagen matrix, elastin fibres and chondrocytes are all heterogeneous on the microscopic scale.Arthritis Research U

Elastic microfibril distribution in the cornea: Differences between normal and keratoconic stroma

The optical and biomechanical properties of the cornea are largely governed by the collagen-rich stroma, a layer that represents approximately 90% of the total thickness. Within the stroma, the specific arrangement of superimposed lamellae provides the tissue with tensile strength, whilst the spatial arrangement of individual collagen fibrils within the lamellae confers transparency. In keratoconus, this precise stromal arrangement is lost, resulting in ectasia and visual impairment. In the normal cornea, we previously characterised the three-dimensional arrangement of an elastic fiber network spanning the posterior stroma from limbus-to-limbus. In the peripheral cornea/limbus there are elastin-containing sheets or broad fibers, most of which become microfibril bundles (MBs) with little or no elastin component when reaching the central cornea. The purpose of the current study was to compare this network with the elastic fiber distribution in post-surgical keratoconic corneal buttons, using serial block face scanning electron microscopy and transmission electron microscopy. We have demonstrated that the MB distribution is very different in keratoconus. MBs are absent from a region of stroma anterior to Descemet's membrane, an area that is densely populated in normal cornea, whilst being concentrated below the epithelium, an area in which they are absent in normal cornea. We contend that these latter microfibrils are produced as a biomechanical response to provide additional strength to the anterior stroma in order to prevent tissue rupture at the apex of the cone. A lack of MBs anterior to Descemet's membrane in keratoconus would alter the biomechanical properties of the tissue, potentially contributing to the pathogenesis of the disease

Non uniformity: structural strategy for optimizing functionality in skeletal ligaments

Ligaments serve as compliant connectors between hard tissues. In that role, they function under various load regimes and directions. However, the 3D structure of ligaments is still considered uniform. The periodontal ligament connects the tooth to the bone and like other ligaments, it sustains different types of loads in various directions. Using the PDL as a model, and employing a fabricated motorized set-up in a microCT instrument, morphological automated segmentation methods and 2nd harmonic generation imaging, we demonstrate that the fibrous network structure within the PDL is not uniform, even before the tooth becomes functional. We find that areas sustaining compression loads are pre-structured with sparse collagenous networks and large blood vessels, whereas other areas contain dense collagen networks with few blood vessels. Therefore, the PDL develops as a non-uniform structure, with an architecture designed to sustain specific types of load in different areas. Based on these findings, we propose that ligaments in general should be regarded as non-uniform entities structured for optimal functioning under variable load regimes.2019-09-26T00:00:00

Elastin is responsible for the rigidity of the ligament under shear and rotational stress : a mathematical simulation study

Background An accurate understanding of the mechanical response of ligaments is important for preventing their damage and rupture. To date, ligament mechanical responses are being primarily evaluated using simulations. However, many mathematical simulations construct models of uniform fibre bundles or sheets using merely collagen fibres and ignore the mechanical properties of other components such as elastin and crosslinkers. Here, we evaluated the effect of elastin-specific mechanical properties and content on the mechanical response of ligaments to stress using a simple mathematical model. Methods Based on multiphoton microscopic images of porcine knee collateral ligaments, we constructed a simple mathematical simulation model that individually includes the mechanical properties of collagen fibres and elastin (fibre model) and compared with another model that considers the ligament as a single sheet (sheet model). We also evaluated the mechanical response of the fibre model as a function of the elastin content, from 0 to 33.5%. Both ends of the ligament were fixed to a bone, and tensile, shear, and rotational stresses were applied to one of the bones to evaluate the magnitude and distribution of the stress applied to the collagen and elastin at each load. Results Uniform stress was applied to the entire ligament in the sheet model, whereas in the fibre model, strong stress was applied at the junction between collagen fibres and elastin. Even in the same fibre model, as the elastin content increased from 0 to 14.4%, the maximum stress and displacement applied to the collagen fibres during shear stress decreased by 65% and 89%, respectively. The slope of the stress–strain relationship at 14.4% elastin was 6.5 times greater under shear stress than that of the model with 0% elastin. A positive correlation was found between the stress required to rotate the bones at both ends of the ligament at the same angle and elastin content. Conclusions The fibre model, which includes the mechanical properties of elastin, can provide a more precise evaluation of the stress distribution and mechanical response. Elastin is responsible for ligament rigidity during shear and rotational stress

Monitoring wound healing of elastic cartilage using multiphoton microscopy

SummaryObjectiveTo demonstrate the ability of multiphoton microscopy (MPM) for monitoring wound healing of elastic cartilage.MethodIn a rabbit ear model, four cartilage specimen groups at 1-day, 1-, 4-, 20-week healing time points as well as a normal elastic cartilage were examined with MPM without using labeling agents. MPM images at wound margins were obtained from specimens at different healing stages, compared with the Hematoxylin and Eosin (H&E) stained images. Image analysis was performed to characterize the collagen morphology for quantifying the wound healing progression of elastic cartilage.ResultsMPM provided high-resolution images of elastic cartilage at varying depths. Comparisons of the images of specimens at different healing stages show obvious cell growth and matrix deposition. The results are consistent with the histological results. Moreover, quantitative analysis results show significant alteration in the collagen cavity size or collagen orientation index during wound healing of elastic cartilage, indicating the possibility to act as indicators for monitoring wound healing.ConclusionOur results suggested that MPM has the ability to monitor the wound healing progression of elastic cartilage, based on the visualization of cell growth and proliferation and quantitative characterization of collagen morphology during wound healing