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. 2021 Aug;102(4-5):218-227.
doi: 10.1111/iep.12399. Epub 2021 Oct 6.

A simple optical tissue clearing pipeline for 3D vasculature imaging of the mediastinal organs in mice

Quanchao Sun  1   2   3 , Picascia Tiziana  2   3 , Arif Ul Maula Khan  2   3 , Vincent Heuveline  4 , Norbert Gretz  2   3
Affiliations

A simple optical tissue clearing pipeline for 3D vasculature imaging of the mediastinal organs in mice

Quanchao Sun et al. Int J Exp Pathol. 2021 Aug.

Abstract

Optical tissue clearing (OTC) methods render tissue transparent by matching the refractive index within a sample to enable three-dimensional (3D) imaging with advanced microscopes. The application of OTC method in mediastinal organs in mice remains poorly understand. Our aim was to establish a simple protocol pipeline for 3D imaging of the mediastinal organs in mice. Trachea, oesophagus, thymus and heart were harvested from mice after retrograde perfusion via the abdominal aorta. We combined and optimized antibody labelling of thick tissue samples, OTC with cheap and non-toxic solvent ethyl cinnamate (ECi), and light-sheet fluorescence microscopy (LSFM) or laser confocal fluorescence microscopy (LCFM) to visualize the vasculature of those tissues. A high degree of optical transparency of trachea, oesophagus, thymus and heart was achieved after ECi-based OTC. With anti-CD31 antibody immunofluorescence labelling before ECi-based OTC, the vasculature of these tissues with their natural morphology, location and organizational network was imaged using LSFM or LCFM. This simple protocol pipeline provides an easy-to-setup and comprehensive way to study the vasculature of mediastinal organs in 3D without any special equipment. We anticipate that it will facilitate diverse applications in biomedical research of thoracic diseases and even other organs.

Keywords: 3D imaging; ethyl cinnamate; mediastinal organs; optical tissue clearing; vasculature.

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Conflict of interest statement

The authors declared that there are no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Whole mediastinal organs OTC and imaging workflow. A, Schematic representation of the proposed research schedule: 1, Organs were extracted following the retrograde perfusion with saline/heparin and 4% PFA via the abdominal aorta. 2, Samples were immunolabelled through the steps of permeabilization and blocking, antibody staining and washing. After that, the stained samples were fixed in 4% PFA again. 3, Tissues were treated with graded concentrations of ethanol for dehydration and optically cleared by ECi for RI matching. 4, Samples were mounted and scanned using LSFM or LCFM by taking sequential z‐stacks through the thick tissue. B, Representative macroscopic images of the mediastinal organs after fixation or ECi‐based OTC. The size of the squares is 5 mm
FIGURE 2
FIGURE 2
Representative images of the vasculature of trachea after immunolabelling and ECi‐based OTC using LSFM, 5× objective. A, 3D reconstruction of trachea based on autofluorescence (green) and vasculature labelled with anti‐CD31 antibody immunostaining (red) as the white arrow indicated. B, Inset of A was zoomed in to show details of anti‐CD31 antibody‐stained vasculature (white arrow) of trachea. C, Depth coding image of the fluorescence‐labelled vasculature of trachea. D‐E, 2D sectional views at different focal planes of the inset from A. The arrows indicate the section of vessel. Scale bar: 300 μm
FIGURE 3
FIGURE 3
Representative images of the vasculature of oesophagus after immunolabelling and ECi‐based OTC using LSFM, 5× objective. A, 3D reconstruction of oesophagus based on autofluorescence (green) and vasculature labelled with anti‐CD31 antibody immunostaining (red) as the white arrow indicated. B, Inset of A in a higher magnification, showing details of anti‐CD31 antibody‐stained vasculature (white arrow) of oesophagus. C, Depth coding image of the fluorescence‐labelled vasculature of oesophagus. D, Cross section of the 3D image of oesophagus shows the vasculature (white arrow) from lumen. E, 2D sectional view at one focal plane of the inset from A. The arrows indicate the section of vessel. Scale bar: 200 μm
FIGURE 4
FIGURE 4
Representative images of the vasculature of thymus after immunolabelling and ECi‐based OTC using LCFM, 16× objective. A, 3D reconstruction of thymus based on autofluorescence (green) and vasculature labelled with anti‐CD31 antibody immunostaining (red) as the white arrow indicated. B, Inset of A in a higher magnification, showing details of anti‐CD31 antibody‐stained vasculature (white arrow) of thymus. C, Depth coding image of the fluorescence‐labelled vasculature of thymus. D, 2D sectional view at one focal plane of the inset from A. The arrow indicates the section of vessel. Scale bar: 300 μm
FIGURE 5
FIGURE 5
Representative images of the vasculature of heart after immunolabelling and ECi‐based OTC using LCFM, 16× objective. A, 3D reconstruction of the heart wall based on autofluorescence (green) and vessels labelled with anti‐CD31 antibody immunostaining (red) as the white arrow indicated. B, Zoomed‐in view of inset area of A shows anti‐CD31 antibody‐stained vasculature (white arrow) of heart in detail. C, Depth coding image of the fluorescence‐labelled vasculature of heart. D, 2D sectional view at a focal plane of the inset from A. The arrow indicates the section of vessel. Scale bar: 300 μm
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