Improved three-dimensional Fourier domain optical coherence tomography by index matching in alveolar structures

Three-dimensional Fourier domain optical coherence tomography (3-D FDOCT) is used to demonstrate that perfusion fixation with a mixture of glutaraldehyde and paraformaldehyde does not alter the geometry of subpleural lung parenchyma in isolated and perfused rabbit lungs. This is confirmed by simultaneous imaging of lung parenchyma with intravital microscopy. To eliminate the diffraction index interfaces between alveolar pockets and walls, we fill the fixed lungs with ethanol by perfusing with gradually increasing concentrations. This bottom-up filling process leaves no remaining air bubbles in the alveolar structures, thus drastically improving the resolution and penetration depth of 3-D FDOCT imaging. We observe an approximately 18% increase in alveolar area after ethanol filling, likely due in large part to elimination of the air/tissue interfaces. 3-D OCT datasets acquired from ethanol-filled lungs allow segmentation of the ethanol-filled structures, which were formerly air-filled, and 3-D reconstruction of larger areas of subpleural alveolar structures. Our innovative process of filling the lungs with ethanol postperfusion fixation thus enables more accurate quantification of alveolar geometries, a critical component of modeling lung function.