Clearing for Deep Tissue Imaging

Abstract

Biologic tissues are generally opaque due to optical properties that result in scattering and absorption of light. Preparation of tissues for optical microscopy often involves sectioning to a thickness of 50-100 µm, the practical limits of light penetration and recovery. A researcher who wishes to image a whole tissue must acquire potentially hundreds of individual sections before rendering them into a three-dimensional volume. Clearing removes strongly light-scattering and light-absorbing components of a tissue and equalizes the refractive index of the imaging medium to that of the tissue. After clearing, the maximum depth of imaging is often defined by the microscope optics rather than the tissue. Such visibility enables the interrogation of whole tissues and even animals without the need to section. Researchers can study a biological process in the context of its three-dimensional environment, identify rare events in large volumes of tissues, and trace cells and cell-cell interactions over large distances. This article describes four popular clearing protocols that are relevant to a wide variety of scenarios across biologic disciplines: CUBIC, CLARITY, 3DISCO, and SeeDB. © 2018 by John Wiley & Sons, Inc.

Purine nucleoside phosphorylase inhibition ameliorates age-associated lower urinary tract dysfunctions

“…Chronic treatment of aged rats with 8-AG ( Figure 3C ) reduced the number of tortuous vessels, and the tissue no longer appeared ischemic, resembling young tissue. Prior to imaging, bladders were cleared using the CUBIC method ( 18 ), and images were acquired by ribbon-scanning confocal microscopy ( 19 ). In addition, as shown in Figure 3D , compared with younger rats, untreated aged rats showed a significant decrease in bladder blood flow (measured using a Doppler flowmeter) ( 20 ).…”

“…Bladders that had been perfused transcardially with PBS buffer containing 20 nm yellow-green fluorescent beads (Thermo Fisher Scientific, F8787) to highlight the vasculature were fixed and then cleared by removing lipids and dissolving light-absorbing chromophores within the tissue ( 18 ). Volumetric data were acquired using a Caliber ID RS-G4 ribbon scanning confocal microscope (Caliber ID), which can acquire large-area images at high (350 nm) resolution using a high NA 1.0, 20× long working distance (8mm) lens (CFl190 20xc GLyc; NIKON Inc.; uses a high nA [1.0] long working distance [8 mm] with low magnification [20×]) ( 19 ).…”

Multiphoton Imaging of Collagen, Elastin, and Calcification in Intact Soft‐Tissue Samples

“…However, many tissues, such as the walls of many extracerebral vessels in humans and larger mammals, are thicker than 1 mm and therefore beyond the MPM imaging depth using the approaches described here. Both confocal and MPM imaging depth can be increased by optically clearing the tissue using solutions with refractive indices closer to those of the wall components, reducing light scattering (Muntifering et al., ). Although this process cannot be used on fresh tissue samples, it can increase imaging depths in fixed samples by orders of magnitude.…”

Compensatory hepatic adaptation accompanies permanent absence of intrahepatic biliary network due to YAP1 loss in liver progenitors

“…At this stage, after washing with PBSA, tissue could be stored long-term in PBSA at room temperature or 4C, or they could proceed directly to immunostaining. Tissues were stained as described by Muntifering, et al in 2018 with primary and secondary antibodies. (Muntifering et al, 2018) To ensure even staining throughout the sample, antibodies were applied by using the SWITCH protocol.…”

“…Tissues were stained as described by Muntifering, et al in 2018 with primary and secondary antibodies. (Muntifering et al, 2018) To ensure even staining throughout the sample, antibodies were applied by using the SWITCH protocol. (Murray et al, 2015) All incubations took place at 30C.…”