Quality and functionality of engineered tissues are closely related to the microstructures and integrity of their extracellular matrix (ECM). However, currently available methods for characterizing ECM structures are often labor-intensive, destructive, and limited to a small fraction of the total area. These methods are also inappropriate for assessing temporal variations in ECM structures. In this study, to overcome these limitations and challenges, we propose an elastic light scattering approach to spatiotemporally assess ECM microstructures in a relatively large area in a nondestructive manner. To demonstrate its feasibility, we analyze spectroscopic imaging data obtained from acellular collagen scaffolds and dermal equivalents as model ECM structures. For spatial characterization, acellular scaffolds are examined after a freeze/thaw process mimicking a cryopreservation procedure to quantify freezing-induced structural changes in the collagen matrix. We further analyze spatial and temporal changes in ECM structures during celldriven compaction in dermal equivalents. The results show that spectral dependence of light elastically backscattered from engineered tissue is sensitively associated with alterations in ECM microstructures. In particular, a spectral decay rate over the wavelength can serve as an indicator for the pore size changes in ECM structures, which are at nanometer scale. A decrease in the spectral decay rate suggests enlarged pore sizes of ECM structures. The combination of this approach with a whole-field imaging platform further allows visualization of spatial heterogeneity of EMC microstructures in engineered tissues. This demonstrates the feasibility of the proposed method that nano- and micrometer scale alteration of the ECM structure can be detected and visualized at a whole-field level. Thus, we envision that this spectroscopic imaging approach could potentially serve as an effective characterization tool to nondestructively, accurately, and rapidly quantify ECM microstructures in engineered tissue in a large area. Copyright © 2013 by ASME.