Optical clearing: impact of optical and dielectric properties of clearing solutions on pulmonary tissue mechanics

Optical clearing allows tissue visualization under preservation of organ integrity. Optical clearing of organs with a physiological change in three-dimensional geometry (such as the lung) would additionally allow visualization of macroscopic and microscopic tissue geometry. A prerequisite, however, is the preservation of the native tissue mechanics of the optically cleared lung tissue. We investigated the impact of optical and dielectric properties of clearing solutions on biomechanics and clearing potency in porcine tissue strips of healthy lungs. After fixation, bleaching, and rehydration, four methods of optical clearing were investigated using eight different protocols. The mechanical and optical properties of the cleared lung tissue strips were investigated by uniaxial tensile testing and by analyzing optical transparency and translucency for red, green, and blue light before, during, and after the biochemical optical clearing process. Fresh tissue strips were used as controls. Best balance between efficient clearing and preserved mechanics was found for clearing with a 1:1 mixture of dimethyl sulfoxide (DMSO) and aniline. Our findings show that 1) the degree of tissue transparency and translucency correlated with the refractive index of the clearing solution index (r = 0.976, P = 0.0004; and r = 0.91, P = 0.0046, respectively), 2) tissue mechanics were affected by dehydration and the type of clearing solution, and 3) tissue biomechanics and geometry correlated with the dielectric constant of the clearing solution (r = -0.98, P < 0.00001; and r = 0.69, P = 0.013, respectively). We show that the lower the dielectric constant of the clearing solutions, the larger the effect on tissue stiffness. This suggests that the dielectric constant is an important measure in determining the effect of a clearing solution on lung tissue biomechanics. Optimal tissue transparency requires complete tissue dehydration and a refractive index of 1.55 of the clearing solution.NEW & NOTEWORTHY Investigating optical clearing in porcine lung tissue strips, we found that refractive index and dielectric constant of the clearing solution affected tissue clearing and biomechanics. By documenting the impact of the composition of the clearing solution on clearing potency and preservation of tissue mechanics, our results help to compose optimal clearing solutions. In addition, the results allow conclusions on the molecular interaction of solvents with collagen fibers in tissue, thereby consolidating existing theories about the functionality of collagen.