European Light Microscopy Initiative Meeting 2022

Abstract

Generation of a 3D cellular topography of biological specimens is often essential to understand cellular and molecular processes. For a long time, biologists simply accepted the time consuming work of preparing sections, which was limited to 2D and often affected by artifacts like ripping and folding. In the last decade, clearing methods in combination with light-sheet fluorescence microscopy emerged as powerful tools for biological imaging in 3D. However, most clearing techniques are based on toxic organic solvents which require special safety provisions and generate extra effort for sample preparation as well as imaging. 

To overcome these shortcomings, we developed a novel clearing procedure based on non-toxic reagents allowing for efficient clearing of various adult mouse organs, like brain hemispheres, tumors, spheroids and organoids. Toward 3D imaging, we combined our clearing procedure with whole-mount immunostaining using Miltenyi's recombinant REAfinity™ antibodies coupled to newly developed Vio® dye variants optimized for application in light-sheet microscopy. Our data indicated highly efficient, bright and stable labelling of various structures in 3D tissue. After data acquisition, our in-house developed post processing pipeline that enables the elimination of intensity inhomogeneities and deconvolution. This allows the ideal preparation of the 3D data for subsequent data visualization and quantification. 

In summary, we developed a robust, fast, and non-toxic clearing procedure combined with bright antibody conjugates for optimal visualization of tissue in 3D, paving the way for a better understanding of tissue structures and disease mechanisms.