Research Techniques Made Simple: Optical Clearing and Three-Dimensional Volumetric Imaging of Skin Biopsies

Abstract

Skin histology is traditionally carried out using two-dimensional tissue sections, which allows for rapid staining, but these sections cannot accurately represent three-dimensional structures in skin such as nerves, vasculature, hair follicles, and sebaceous glands. Although it may be ideal to image skin in a three-dimensional manner, it is technically challenging to image deep into tissue because of light scattering from collagen fibrils in the dermis and refractive index mismatch owing to the presence of differing biological materials such as cytoplasm, and lipids in the skin. Different optical clearing methods have been developed recently, making it possible to render tissues transparent using different approaches. Here, we discuss the steps involved in tissue preparation for three-dimensional volumetric imaging and provide a brief overview of the different optical clearing methods as well as different imaging modalities for three-dimensional imaging.

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“…Furthermore, optical clearing protocols can be broadly divided into aqueous or organic solvent‐based protocols, and numerous protocols have been developed over the past decade due to ongoing interest to visualize whole organs. As it is beyond the scope of this review to cover the different protocols, we would like to refer the readers to the following excellent reviews on the topic 161‐163 and a review addressing skin optical clearing 164 …”

“…Furthermore, optical clearing protocols can be broadly divided into aqueous or organic solvent-based protocols, and numerous protocols have been developed over the past decade due to ongoing interest to visualize whole organs. As it is beyond the scope of this review to cover the different protocols, we would like to refer the readers to the following excellent reviews on the topic[161][162][163] and a review addressing skin optical clearing 164. Imaging of whole organs has been aided by the development of light sheet microscopes (LSM) which use a sheet of light to sequentially illuminate a single plane within the tissue, enabling rapid 3D volumetric imaging of the entire organ.…”