The biophysical and biochemical properties of live tissues are important in the context of development and disease. Methods for evaluating these properties typically involve destroying the tissue or require specialized technology and complicated analyses. Here, we present a novel, noninvasive methodology for determining the spatial distribution of tissue features within embryos, making use of nondirectionally migrating cells and software we termed "Landscape," which performs automatized high-throughput three-dimensional image registration. Using the live migrating cells as bioprobes, we identified structures within the zebrafish embryo that affect the distribution of the cells and studied one such structure constituting a physical barrier, which, in turn, influences amoeboid cell polarity. Overall, this work provides a unique approach for detecting tissue properties without interfering with animal's development. In addition, Landscape allows for integrating data from multiple samples, providing detailed and reliable quantitative evaluation of variable biological phenotypes in different organisms.