Differential interference contrast and confocal reflectance imaging of collagen organization in three‐dimensional matrices

The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing as well as in tissue engineering. Three‐dimensional (3‐D) type I collagen matrices have been used extensively as an in vitro model for studying cell‐induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the process of 3‐D extracellular matrix (ECM) remodeling at the cellular and subcellular level. In this study, we directly compare two imaging modalities for both quantitative and qualitative imaging of 3‐D collagen organization in vitro: differential interference contrast (DIC) and confocal reflectance imaging. The results demonstrate that two‐dimensional (2‐D) DIC images allow visualization of the same population of collagen fibrils as observed in 2‐D confocal reflectance images. Thus, DIC can be used for qualitative assessment of fibril organization, as well as tracking of fibril movement in sequential time‐lapse 2‐D images. However, we also found that quantitative techniques that can be applied to confocal reflectance images, such as Fourier transform analysis, give different results when applied to DIC images. Furthermore, common techniques used for 3‐D visualization and reconstruction of confocal reflectance datasets are not generally applicable to DIC. Overall, obtaining a complete understanding of cell‐matrix mechanical interactions will likely require a combination of both wide‐field DIC imaging to study rapid changes in ECM deformation which can occur within minutes, and confocal reflectance imaging to assess more gradual changes in cell‐induced compaction and alignment of ECM which occur over a longer time course.