Tissue optical clearing technique provides a prospective solution for the application of advanced optical methods in life sciences. This paper gives a review of recent developments in tissue optical clearing techniques. The physical, molecular and physiological mechanisms of tissue optical clearing are overviewed and discussed. Various methods for enhancing penetration of optical‐clearing agents into tissue, such as physical methods, chemical‐penetration enhancers and combination of physical and chemical methods are introduced. Combining the tissue optical clearing technique with advanced microscopy image or labeling technique, applications for 3D microstructure of whole tissues such as brain and central nervous system with unprecedented resolution are demonstrated. Moreover, the difference in diffusion and/or clearing ability of selected agents in healthy versus pathological tissues can provide a highly sensitive indicator of the tissue health/pathology condition. Finally, recent advances in optical clearing of soft or hard tissue for in vivo imaging and phototherapy are introduced.

We developed an advanced clearing method with superior fluorescence-preserving capability for 3D imaging of whole organs.

Advanced optical methods combined with various probes pave the way toward molecular imaging within living cells. However, major challenges are associated with the need to enhance the imaging resolution even further to the subcellular level for the imaging of larger tissues, as well as for in vivo studies. High scattering and absorption of opaque tissues limit the penetration of light into deep tissues and thus the optical imaging depth. Tissue optical clearing technique provides an innovative way to perform deep-tissue imaging. Recently, various optical clearing methods have been developed, which provide tissue clearing based on similar physical principles via different chemical approaches. Here, we introduce the mechanisms of the current clearing methods from fundamental physical and chemical perspectives, including the main physical principle, refractive index matching via various chemical approaches, such as dissociation of collagen, delipidation, decalcification, dehydration, and hyperhydration, to reduce scattering, as well as decolorization to reduce absorption.

Imaging cells and microvasculature in the living brain is crucial to understanding an array of neurobiological phenomena. Here, we introduce a skull optical clearing window for imaging cortical structures at synaptic resolution. Combined with two-photon microscopy, this technique allowed us to repeatedly image neurons, microglia and microvasculature of mice. We applied it to study the plasticity of dendritic spines in critical periods and to visualize dendrites and microglia after laser ablation. Given its easy handling and safety, this method holds great promise for application in neuroscience research.

Tissue optical clearing techniques have provided important tools for large‐volume imaging. Aqueous‐based clearing methods are known for good fluorescence preservation and scalable size maintenance, but are limited by long incubation time, insufficient clearing performance, or requirements for specialized devices. Additionally, few clearing methods are compatible with widely used lipophilic dyes while maintaining high clearing performance. Here, to address these issues, m‐xylylenediamine (MXDA) is firstly introduced into tissue clearing and used to develop a rapid, highly efficient aqueous clearing method with robust lipophilic dyes compatibility, termed MXDA‐based Aqueous Clearing System (MACS). MACS can render whole adult brains highly transparent within 2.5 days and is also applicable for other intact organs. Meanwhile, MACS possesses ideal compatibility with multiple probes, especially for lipophilic dyes. MACS achieves 3D imaging of the intact neural structures labeled by various techniques. Combining MACS with DiI labeling, MACS allows reconstruction of the detailed vascular structures of various organs and generates 3D pathology of glomeruli tufts in healthy and diabetic kidneys. Therefore, MACS provides a useful method for 3D mapping of intact tissues and is expected to facilitate morphological, physiological, and pathological studies of various organs.