Microscale heterogeneities in tissue rheological properties such as stiffness and viscosity strongly influence cell fate and malignancy. However, outstanding questions about the timescales of interactions (measured as a range of frequencies), length scales and type of interactions sensed by cells within tissues that are physiologically relevant remain unanswered. What is needed is the ability to resolve and quantitate minute forces that cells sense in the local environment (on the order of microns) within thick tissue (~mm) and 3D culture models, that approximate clinically relevant in vivo architecture and signaling cues, allowing for real time characterization of cell-ECM dynamics. We performed active Microrheology using an Optical Trap In Vivo (aMOTIV) microscopy using an in situ calibration method to obtain exact trap stiffness at each probe to quantify local applied forces with high spatial and temporal resolutions. This allowed us to determine tissue mechanics at length scales (nm-mm) and frequencies (1-10,000’s Hz) unobtainable by bulk rheology, which misses the cell-scale heterogeneities, or with passive microrheology, which misses the interesting non-linear stress/strain curves seen with active probing then apply defined strains, Applicable to thick tissue, this technique allowed us to distinguish mechanical heterogeneities with micrometer spatial resolution at penetration depths up to 500 m. After initial characterization of 3D cell culture gels, we applied our technique in zebrafish, Danio rerio; the first time in situ calibration and microrheology has been applied to a living model vertebrate organism. Our initial data indicates a broad range of elastic moduli, with measurements in the tail ranging ~ 10s to 1000s of Pascals, while the brain is significantly softer (10’s to 100’s Pa). We show here an advanced technique for micro-rheological characterization in vitro and in vivo, to accurately quantify physical determinants of the local microenvironment. This allows for accurate measurements mechanical properties in living organisms and in tissue models to definitively account for microenvironmental impact on individual cells and organogenesis.