Second Harmonic and Brillouin imaging to probe vocal folds' biomechanics – HaBIm

Mainly composed of fibrillar collagen, highly organized at different hierarchical scales, connective tissues (e.g. cartilage, skin, bone, arteries, cornea) provides structural scaffold to many organs and determine the micro-environment properties influencing cell adhesion and migration. Despite common constituents, these tissues present widely different mechanical behaviors and functions. It exists extensive literature dealing with the mechanical characterization of individual collagen fibrils and with the assessment of tissues’ macroscopic response. Yet, the interplay between these scale is left obscure, mostly due to the lack of appropriate techniques to probe them simultaneously. For example, the origin of the highly nonlinear strain-dependent stiffening of collagen network remains unknown, let alone how these macroscopic behaviors arise from the micro-architecture of the tissue. In this context, deciphering the complex role of collagen network organization in the formation of macroscopic biomechanical properties of living tissues is of utmost interest, since they play an intricate role in determining physio-pathological behavior of these tissues.
Over the years, Second Harmonic Generation (SHG) microscopy has imposed as the gold standard for collagen imaging in live thick tissues, enabling label-free visualization of fibrillar distribution, with high intrinsic specificity and sub-micron spatial resolution. In parallel, the recent advent of Brillouin microscopy has revolutionized the field of biomechanics, allowing contact-less and non-invasive mapping of elastic properties, with sub-micron resolution, in soft and heterogeneous medium.
Leveraging these recent advances HaBIm seeks to implement an innovative microscope, coupling state-of-the-art SHG and Brillouin imaging while enabling simultaneous mechanical assay under the microscope. Pairing macroscopic stress/strain measures, microscopic viscoelasticity probing and nanoscopic tissue reorganization, this cutting-edge instrument will provide new insights into the multiscale relationship between collagen architecture and mechanical behavior. Moreover, a Raman spectrometer, enabling to map the chemical content in the sample, will be added to contextualize the complementary information. Upon operational, this platform will be validated on mouse vocal folds at various macroscopic deformation, to characterize the collagen organization and viscoelastic properties in depth in the different layers of the tissue, from epithelium to the vocalis muscle, through the lamina propria. As a proof-of-concept, monitoring fine changes after laser-induced lesions will demonstrate the potential of this method to characterize local alteration of the tissue at microscopic scale and the resulting defective mechanical properties.
Lying at the interface between advanced optical microscopy and biomechanics of connective tissues, HaBIm aims to close the instrumental gap between SHG imaging and Brillouin microscopy to correlate multiscale mechanical measurements and structural imaging. Associated with advanced image analysis approaches, this project will pave the way for a complete mechanical characterization of connective tissues. Going beyond the state-of-the-art, this project will establish an analytical framework to bridge the multiple scale involved in the complex morpho-functional relationship of live tissues. Such methodology holds the potential to unveil new insights into the crucial role of viscoelastic properties in determining physiological (e.g. development, aging) and pathological behaviors (e.g. genetic diseases, wound healing). Potential applications include monitoring early-stage alteration in the context of tumor progression and metastatic invasion. Moreover, this approaches could guide the design and validation of biomimetic sample used in tissue engineering.