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  • Multispectral confocal 3D imaging of intact healthy and tumor tissue using mLSR-3D.

Multispectral confocal 3D imaging of intact healthy and tumor tissue using mLSR-3D.

Nature protocols (2022-10-01)
Ravian L van Ineveld, Raphaël Collot, Mario Barrera Román, Anna Pagliaro, Nils Bessler, Hendrikus C R Ariese, Michiel Kleinnijenhuis, Marcel Kool, Maria Alieva, Susana M Chuva de Sousa Lopes, Ellen J Wehrens, Anne C Rios
ABSTRACT

Revealing the 3D composition of intact tissue specimens is essential for understanding cell and organ biology in health and disease. State-of-the-art 3D microscopy techniques aim to capture tissue volumes on an ever-increasing scale, while also retaining sufficient resolution for single-cell analysis. Furthermore, spatial profiling through multi-marker imaging is fast developing, providing more context and better distinction between cell types. Following these lines of technological advance, we here present a protocol based on FUnGI (fructose, urea and glycerol clearing solution for imaging) optical clearing of tissue before multispectral large-scale single-cell resolution 3D (mLSR-3D) imaging, which implements 'on-the-fly' linear unmixing of up to eight fluorophores during a single acquisition. Our protocol removes the need for repetitive illumination, thereby allowing larger volumes to be scanned with better image quality in less time, also reducing photo-bleaching and file size. To aid in the design of multiplex antibody panels, we provide a fast and manageable intensity equalization assay with automated analysis to design a combination of markers with balanced intensities suitable for mLSR-3D. We demonstrate effective mLSR-3D imaging of various tissues, including patient-derived organoids and xenografted tumors, and, furthermore, describe an optimized workflow for mLSR-3D imaging of formalin-fixed paraffin-embedded samples. Finally, we provide essential steps for 3D image data processing, including shading correction that does not require pre-acquired shading references and 3D inhomogeneity correction to correct fluorescence artefacts often afflicting 3D datasets. Together, this provides a one-week protocol for eight-fluorescent-marker 3D visualization and exploration of intact tissue of various origins at single-cell resolution.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-Vimentin Antibody, clone VIM 3B4, clone VIM 3B4, Chemicon®, from mouse
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, essentially globulin free, pH 7, ≥98%
Sigma-Aldrich
Anti-Olig-2 Antibody Alexa Fluor 555 Conjugate, from rabbit, ALEXA FLUOR 555
Sigma-Aldrich
Anti-Olig-2 Antibody, Chemicon®, from rabbit