Project Summary/Abstract The goal of this proposal is to develop and validate a laser SpeckLe fIeld Microrheology (SLIM) technology for micromechanical mapping of the tissue ECM, with lateral resolution of 10 μm, axial resolution of 60 μm, and a penetration depth of 5 mm penetration depth. ECM stiffness, as perceived by cells, is emerging as a prominent micro-mechanical cue that precedes pathogenesis and directs its progression by orchestrating nearly all aspects of cellular behavior. Excessive and irregular micro-mechanical remodeling of the ECM is implicated in a broad spectrum of pathologies, including cardiovascular disease, fibrotic disorders, and cancer, which together account for over 50% of death worldwide. Nevertheless, our understanding of the underlying mechanisms is severely limited as currently there are no imaging tools available for micromechanical mapping of the ECM at length scales pertinent to cells. SLIM measures the time-varying speckle intensity fluctuations. Speckle is a grainy intensity pattern, formed when a coherent laser beam is back scattered from tissue. Brownian displacements of scattering particles within the ECM dynamically modulate the speckle fluctuations. These fluctuations in turn are intimately related to the viscoelastic properties of imaged tissue. In compliant regions, unrestricted Brownian displacements provoke rapidly fluctuating speckle spots, whereas in rigid areas, restrained motions elicit limited intensity variations of speckle grains. Pixel-wise correlation analysis of intensity fluctuations provides a 2D depth-integrated map of mechanical properties within the tissue. However, the resolution of this map is limited to the speckle grain size, set by the Numerical Aperture (NA) of optics. In addition, due to multiple scattering of light, speckle fluctuations are modulated by the Brownian displacements of the scattering particles within the entire illuminated volume. As a result, the evaluated map lacks depth information. Therefore, the first goal of this proposal is to address these issues by introducing an innovative SLIM platform, capable of high resolution, depth-resolved, large FoV, micromechanical mapping of the ECM, without physical scanning and refocusing on the sample. Our second goal is then to identify the link between the micromechanical properties of ECM and known hallmarks of disease progression, by focusing on breast cancer as a model. The unique capability of SLIM for micro-mechanical tomography of ECM enables identifying the key biomechanical mediators of pathogenies. It also opens multiple avenues based on targeting the cell-ECM micromechanical interactions for therapeutic management of disease.