Statistical characterization of engineered tissues using confocal mosaic technology

Characterization of engineered tissues using optical methods often involves tradeoff between the fraction of total volume that is imaged and the spatial resolution. The limitation is not technological but rather practical, having more to do with effective probe designs and computer memory storage for large datasets. In this paper, we propose using confocal mosaicing, a technique used to characterize large volumes of excisioned biopsies from Mohs surgeries, to characterizing collagen gels. This technique stitches together high-resolution 3D images of a series adjacent millimeter sized regions that collectively make up areas that are ∼cm². Image acquisition time is approximately 5 min. The resulting high-resolution images closely resemble hematoxylin and eosin histological sections, only obtained without the time-consuming embedding and sectioning steps. Disk-shaped collagen gels that are 1 ml volume and ~1.5 cm diameter were prepared with smooth muscle cells and imaged at days 1 and 5. Using the digital staining technique, we were able to survey the spatial distribution of cells in the hydrogel and assess spatial heterogeneity in 3D from the fluorescence data. The reflectance data provided information on collagen fibril structure and matrix remodeling by the cells. Digital staining presented the data in a way that is easily interpreted by tissue engineers. Altogether, we believe confocal mosaicing and digital staining represents an important technological novelty that significantly advances nondestructive optical evaluation of engineered tissues.