From 2D to 3D: Promising Advances in Imaging Lung Structure

Abstract

The delicate structure of murine lungs poses many challenges for acquiring high-quality images that truly represent the living lung. Here, we describe several optimized procedures for obtaining and imaging murine lung tissue. Compared to traditional paraffin cross-section and optimal cutting temperature (OCT), agarose-inflated vibratome sections (aka precision-cut lung slices), combines comparable structural preservation with experimental flexibility. In particular, we discuss an optimized procedure to precision-cut lung slices that can be used to visualize three-dimensional cell-cell interactions beyond the limitations of two-dimensional imaging. Super-resolution microscopy can then be used to reveal the fine structure of lung tissue's cellular bodies and processes that regular confocal cannot. Lastly, we evaluate the entire lung vasculature with clearing technology that allows imaging of the entire volume of the lung without sectioning. In this manuscript, we combine the above procedures to create a novel and evolutionary method to study cell behavior ex vivo , trace and reconstruct pulmonary vasculature, address fundamental questions relevant to a wide variety of vascular disorders, and perceive implications to better imaging clinical tissue.

Lung organoids: advances in generation and 3D-visualization

“…The complexity and delicate structure of the lung are primary reasons why acquiring high-quality microscopy images is difficult, particularly three-dimensional (3D) imaging of the lung for studies that require subcellular resolution while maintaining anatomical structure. Over the last decade, cutting-edge microscopic and quantitative histological techniques have been developed for 3D imaging of lung tissue (reviewed in (Schittny 2018)) and, although innovations have been made with regard to lung tissue clearing and preparation, the acquisition of highquality, clinically relevant data for 3D imaging remains challenging (Gomez-Gaviro et al 2020;Klouda et al 2020).…”

A pneumonectomy model to study flow-induced pulmonary hypertension and compensatory lung growth

“…Combining deep tissue clearing and advanced 3D imaging of the entire RV using Lightsheet microscopy will better describe the acute and chronic effects on the RV vasculature in flow-induced PH experimental models. 49 …”

Multiresolution 3D Optical Mapping of Immune Cell Infiltrates in Mouse Asthmatic Lung

“…Other challenges in 3D imaging of asthmatic mouse lungs include rendering the tissue sufficiently transparent while maintaining structural integrity and immunogenicity of protein antigens for IF detection of various immune cell types ( 10 , 34 ). Transcardiac perfusion before tissue collection could not effectively remove RBCs in the patchy inflammation regions of asthmatic lungs ( see Figure E2).…”