The effect of wavelength on optical properties extracted from images of engineered tissue

Optical imaging modalities such as confocal microscopy and optical coherence tomography (OCT) are emerging as appealing methods for non-destructive evaluation of engineered tissues. The information offered by such optical imaging methods depends on the wavelength vis-á-vis the optical scattering properties of the sample. These properties affect many factors critical to image analysis in a nonlinear and nontrivial manner. Thus, we sought to characterize the effect wavelength has on the optical properties collagen remodeled by cells at 3 common imaging wavelengths: 488, 633, and 1310 nm. To do this, we seeded smooth muscle cells (SMCs) in soluble collagen gels at a density of 1×106 cells/ml; similar acellular control constructs were also prepared. The constructs were allowed to remodel in the incubator for 5 days, and were examined at 24 and 120 hours by confocal imaging at 488 and 633 nm, and by OCT imaging at 1310 nm. From the confocal and OCT data, the attenuation and reflectivity were evaluated by fitting the data to a theoretical model that relates the tissue optical properties (scattering coefficient and anisotropy factor) and imaging conditions to the signal. In general, we found that at 1310 nm, the optical properties of the acellular control constructs had a lower reflectivity (higher anisotropy) than the SMC constructs. The difference in reflectivity between the SMC construct and acellular controls tended to decrease with wavelength, owing to a relative increase in reflectivity of acellular controls at lower wavelengths relative to the cellular constructs. Overall, the largest difference in optical properties occurred at 1310 nm. Taken together, the data show that the shift in optical properties of soluble collagen gels caused by cellular remodeling is nonlinearly wavelength dependent, and that this information should be considered when devising how to optimally characterize engineered tissues using optical imaging methods.