ADAPT-3D: Accelerated Deep Adaptable Processing of Tissue for 3-Dimensional Fluorescence Tissue Imaging for Research and Clinical Settings

Light sheet microscopy and preparative clearing methods that improve light penetration in 3D tissues have revolutionized imaging in biomedical research. While most clearing methods focus on removing molecules that scatter light, the methods generally involve immersing tissues in solutions that minimize refraction of light to enhance detection of fluorescent signal deeper into tissues. Here, we developed a new tissue preparative method called ADAPT-3D with broad applicability across species and tissue types. This method enables efficient antibody staining and detection of endogenous fluorophores and offers advantages in terms of speed at which tissue staining and clearing is achieved. In about 4 days from tissue harvest to imaging, human intestinal tissue could be fixed, decolored and delipidated to remove light-interfering substances and stained with antibodies for imaging. In the intact mouse skull and brain, involving an 8-day protocol from tissue harvest to completion of imaging, the aqueous and non-shrinking ADAPT-3D method allowed the specialized channels between skull and underlying tissue to be detected without meningeal tearing. Overall, ADAPT-3D provides a highly versatile preparative method for 3D fixed tissue imaging with superior time savings, sensitivity and preservation of tissue morphology compared with previously described methods.