Investigation of changes in optical attenuation of bone and neuronal cells in organ culture or three-dimensional constructs in vitro with optical coherence tomography: relevance to cytochrome oxidase monitoring

Changes in optical attenuation, relevant to cytochrome oxidase, of the rat bone periosteal tissue in explanted culture and human neuronal cells in three-dimensional agarose constructs have been monitored by the use of optical coherence tomography (OCT), with potential applications in tissue engineering and diagnosis. A superluminescent diode (SLD) with a peak emission wavelength (λ=820 nm) that is the near-infrared absorption band of the oxidized form of CytOx was employed. The attenuation coefficient was obtained from the depth-resolved reflectance profiles of liquid phantoms (naphthol green B with intralipid), explant culture (periosteum of calvaria from rats) and cells in 3D agarose constructs. The absorption coefficient of naphthol green B can be accurately quantified by the linear relationship between attenuation coefficients and the concentration. The difference in the attenuation coefficient of astrocytoma cells in agarose before and after reduction of CytOx is 0.26±0.10 mm−1 (n=9), whereas no attenuation is observed with the agarose control. Reduction of the enzyme in periosteal tissue leads to a change in attenuation coefficient of 0.43±0.24 mm−1 (n=7). For comparison, using a biochemical assay, the absorption coefficient of the oxidized-reduced form of CytOx is measured at approximately 8.3±1.5×10−3 mm−1 (n=4) and 8.7±2.5×10−3 mm−1 (n=4) at 820 nm for astrocytoma cells and rat periosteum, respectively. The lower value of CytOx concentration using biochemical versus OCT measurements may result from shifts in the scattering profile and the amplifying influences of multiple heme-based oxidases, indicating that conventional OCT is not specific enough to monitor redox changes in cytochrome oxidase. However, qualitative shifts in oxidation state are apparent using the technique. Our results suggest the potential application of OCT in providing high-resolution tomographic imaging of tissues in organ culture and cells grown in three-dimensional constructs in vitro.