Whole Organ Blood and Lymphatic Vessels Imaging (WOBLI)

Abstract

Thin section histology is limited in providing 3D structural information, particularly of the intricate morphology of the vasculature. Availability of high spatial resolution imaging for thick samples, would overcome the restriction dictated by low light penetration. Our study aimed at optimizing the procedure for efficient and affordable tissue clearing, along with an appropriate immunofluorescence labeling that will be applicable for high resolution imaging of blood and lymphatic vessels. The new procedure, termed whole organ blood and lymphatic vessels imaging (WOBLI), is based on two previously reported methods, CLARITY and ScaleA2. We used this procedure for the analysis of isolated whole ovary, uterus, lung and liver. These organs were subjected to passive clearing, following fixation, immunolabeling and embedding in hydrogel. Cleared specimens were immersed in ScaleA2 solution until transparency was achieved and imaged using light sheet microscopy. We demonstrate that WOBLI allows detailed analysis and generation of structural information of the lymphatic and blood vasculature from thick slices and more importantly, from whole organs. We conclude that WOBLI offers the advantages of morphology and fluorescence preservation with efficient clearing. Furthermore, WOBLI provides a robust, cost-effective method for generation of transparent specimens, allowing high resolution, 3D-imaging of blood and lymphatic vessels networks.

BACH family members regulate angiogenesis and lymphangiogenesis by modulating VEGFC expression

“…We, thus, examined the effects of BACH1 on the lymphatic vasculature in various mouse tumor models. To gain insight into the 3D complexity of the lymphatic network within intact tumors, we subjected ES2 subcutaneous xenografts to a modified CLARITY (Clear, Lipid-exchanged, Anatomically Rigid, Imaging-compatible, Tissue hYdrogel) technique (Hama et al, 2011;Chung et al, 2013;Oren et al, 2018), along with LYVE1 immunofluorescence staining. A robust increase in lymphatic vessel density was observed in BACH1-overexpressing tumors as compared with control-derived ES2 tumors ( Fig 5C and Videos 1 and 2).…”

Advances in Ex Situ Tissue Optical Clearing

“…Oren et al . presented yet another approach for whole organ blood and lymphatic vessels imaging (WOBLI).…”

Recent Advances on Fluorescence Imaging of Lymphatic Vasculature System

“…Through intravital two-photon imaging, researchers realized real-time observation of immune cells in the lymph nodes of the mouse and monitored the interactions between circulating lymphocytes and the stromal barriers. [26] Light-Sheet Fluorescence Microscopy When using thin sections for histological studies, the images are limited in providing 3-D structural information, particularly of the sophisticated morphological information of the vasculature. If thick samples imaging can overcome the limitations of low light penetration, high spatial resolution 3-D imaging of thick samples can be realized.…”