Neuroscience in the third dimension: shedding new light on the brain with tissue clearing

Abstract

For centuries analyses of tissues have depended on sectioning methods. Recent developments of tissue clearing techniques have now opened a segway from studying tissues in 2 dimensions to 3 dimensions. This particular advantage echoes heavily in the field of neuroscience, where in the last several years there has been an active shift towards understanding the complex orchestration of neural circuits. In the past five years, many tissue-clearing protocols have spawned. This is due to varying strength of each clearing protocol to specific applications. However, two main protocols have shown their applicability to a vast number of applications and thus are exponentially being used by a growing number of laboratories. In this review, we focus specifically on two major tissue-clearing method families, derived from the 3DISCO and the CLARITY clearing protocols. Moreover, we provide a “hands-on” description of each tissue clearing protocol and the steps to look out for when deciding to choose a specific tissue clearing protocol. Lastly, we provide perspectives for the development of tissue clearing protocols into the research community in the fields of embryology and cancer.

Multi-Fluorescence High-Resolution Episcopic Microscopy (MF-HREM) for Three-Dimensional Imaging of Adult Murine Organs

“…Where the standard 3D culture could not easily be used, e.g. for in vivo staining routes or specific antibodies, compatibility was tested on cryosections from stained organs (12).…”

“…Three-dimensional (3D) optical microscopy has been the focus of a substantial body of research, with several new technologies coming to prominence in recent years. This has enabled numerous new biological insights to be made in neuroscience, developmental biology, cancer and immunology (2,6,8,12).…”

“…Despite the obvious utility of large-volume, high-resolution 3D optical microscopy, there remain many challenges. Clearing protocols can often be complex and lengthy, and the technicality of serial sectioning instruments as well as the long imaging times limit their widespread adoption (3,12). All of these factors cause delays in optimising new protocols, restricts studies to small group sizes and often require specialised imaging facilities.…”

“…Cleared samples can be imaged using techniques such as light-sheet microscopy (2,4,7) and optical projection tomography (OPT) (10,11). However, clearing requires often complex, lengthy and costly tissue preparation, with variable efficacy (9,12), and widely acknowledged morphological changes to the sample (9,13). Additionally, sample size is limited by the working distance of the microscope objective lens (7) and the objective lens must be protected from the often corrosive clearing solutions.…”

Applicability of vital staining and tissue clearing to vascular anatomy and melanocytes' evaluation of temporal bone in six laboratory species

“…Recently, tissue‐clearing techniques have been developed and used in a variety of research fields due to the introduction of new chemical agents (Moore, Lucas, Goodman, Coolen, & Lehman, ; Togami et al, ; Vigouroux, Belle, & Chédotal, ). During the recent decade, the need for transmitted anatomical images has expanded in biological research fields.…”