Tissue Clearing and Its Application to Bone and Dental Tissues

Opaqueness of animal tissue can be attributed mostly to light absorption and light scattering. In most noncleared tissue samples, confocal images can be acquired at no more than a 100-µm depth. Tissue-clearing techniques have emerged in recent years in the neuroscience field. Many tissue-clearing methods have been developed, and they all follow similar working principles. During the tissue-clearing process, chemical or physical treatments are applied to remove components blocking or scattering the light. Finally, samples are immersed in a designated clearing medium to achieve a uniform refractive index and to gain transparency. Once the transparency is reached, images can be acquired even at several millimeters of depth with high resolution. Tissue clearing has become an essential tool for neuroscientists to investigate the neural connectome or to analyze spatial information of various types of brain cells. Other than neural science research, tissue-clearing techniques also have applications for bone research. Several methods have been developed for clearing bones. Clearing treatment enables 3-dimensional imaging of bones without sectioning and provides important new insights that are difficult or impossible to acquire with conventional approaches. Application of tissue-clearing technique on dental research remains limited. This review will provide an overview of the recent literature related to the methods and application of various tissue-clearing methods. The following aspects will be covered: general principles for the tissue-clearing technique, current available methods for clearing bones and teeth, general principles of 3-dimensional imaging acquisition and data processing, applications of tissue clearing on studying biological processes within bones and teeth, and future directions for 3-dimensional imaging.