Section: New Results

Experimental Assessments

Combination of traction assays and multiphoton imaging to quantify skin biomechanics

Participant : Jean-Marc Allain.

An important issue in tissue biomechanics is to decipher the relationship between the mechanical behavior at macroscopic scale and the organization of the collagen fiber network at microscopic scale. We have formalized a definitive protocol [46] to combine traction assays with multiphoton microscopy in ex vivo murine skin. This multiscale approach provides simultaneously the stress/stretch response of a skin biopsy and the collagen reorganization in the dermis by use of second harmonic generation (SHG) signals and appropriate image processing.

Monitoring dynamic collagen reorganization during skin stretching with fast polarization-resolved second harmonic generation imaging

Participant : Jean-Marc Allain.

The mechanical properties of biological tissues are strongly correlated to the specific distribution of their collagen fibers. Monitoring the dynamic reorganization of the collagen network during mechanical stretching is however a technical challenge, because it requires mapping orientation of collagen fibers in a thick and deforming sample. In this work [24], a fast polarization-resolved second harmonic generation microscope is implemented to map collagen orientation during mechanical assays. This system is based on line-to-line switching of polarization using an electro-optical modulator and works in epi-detection geometry. After proper calibration, it successfully highlights the collagen dynamic alignment along the traction direction in ex vivo murine skin dermis. This microstructure reorganization is quantified by the entropy of the collagen orientation distribution as a function of the stretch ratio. It exhibits a linear behavior, whose slope is measured with a good accuracy. This approach can be generalized to probe a variety of dynamic processes in thick tissues.

Multiscale characterisation of skin mechanics through in-situ imaging

Participant : Jean-Marc Allain.

The complex mechanical properties of skin have been studied intensively over the past decades. They are intrinsically linked to the structure of the skin at several length scales, from the macroscopic layers (epidermis, dermis and hypodermis) down to the microstructural organization at the molecular level. Understanding the link between this microscopic organization and the mechanical properties is of significant interest in the cosmetic and medical fields. Nevertheless, it only recently became possible to directly visualize the skin’s microstructure during mechanical assays, carried out on the whole tissue or on isolated layers. These recent observations have provided novel information on the role of structural components of the skin in its mechanical properties, mainly the collagen fibers in the dermis, while the contribution of others, such as elastin fibers, remains elusive. We performed in [45] a systematic review of the current methods used to observe skin’s microstructure during a mechanical assay, along with their strengths and limitations, as well as a review of the unique information they provide on the link between structure and function of the skin.

Root Hair Sizer: an algorithm for high throughput recovery of different root hair and root developmental parameters

Participant : Jean-Marc Allain.

The root is an important organ for water and nutrient uptake, and soil anchorage. It is equipped with root hairs (RHs) which are elongated structures increasing the exchange surface with the soil. RHs are also studied as a model for plant cellular development, as they represent a single cell with specific and highly regulated polarized elongation. For these reasons, it is useful to be able to accurately quantify RH length employing standardized procedures. Methods commonly employed rely on manual steps and are therefore time consuming and prone to errors, restricting analysis to a short segment of the root tip. Few partially automated methods have been reported to increase measurement efficiency. However, none of the reported methods allow an accurate and standardized definition of the position along the root for RH length measurement, making data comparison difficult. In this work [28] we are developing an image analysis algorithm that semi‐automatically detects RHs and measures their length along the whole differentiation zone of roots. This method, implemented as a simple automated script in ImageJ/Fiji software that we termed Root Hair Sizer, slides a rectangular window along a binarized and straightened image of root tips to estimate the maximal RH length in a given measuring interval. This measure is not affected by heavily bent RHs and any bald spots. RH length data along the root are then modelled with a sigmoidal curve, generating several biologically significant parameters such as RH length, positioning of the root differentiation zone and, under certain conditions, RH growth rate. Image analysis with Root Hair Sizer and subsequent sigmoidal modelling of RH length data provide a simple and efficient way to characterize RH growth in different conditions, equally suitable to small and large scale phenotyping experiments.

Calcium and plasma membrane force-gated ion channels behind development

Participant : Jean-Marc Allain.

During development, tissues are submitted to high variation of compression and tension forces. The roles of the cell wall, the cytoskeleton, the turgor pressure and the cell geometry during this process have received due attention. In contrast, apart from its role in the establishment of turgor pressure, the involvement of the plasma membrane as a transducer of mechanical forces during development has been under studied. Force-gated (FG) or Mechanosensitive (MS) ion channels embedded in the bilayer represent ‘per se’ archetypal mechanosensor able to directly and instantaneously transduce membrane forces into electrical and calcium signals. We reviewed in [26] how their fine-tuning, combined with their ability to detect micro-curvature and local membrane tension, allows FG channels to transduce mechanical cues into developmental signals.