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

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

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)3028-3055
Number of pages28
JournalNature Protocols
Volume17
Issue number12
DOIs
Publication statusPublished - Dec 2022

Keywords

  • Imaging, Three-Dimensional/methods
  • Microscopy, Fluorescence/methods
  • Organoids
  • Microscopy, Confocal/methods

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