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

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.

Temporal morphogen gradient-driven neural induction shapes single expanded neuroepithelium brain organoids with enhanced cortical identity

“…Sections were mounted using Immu-Mount mounting medium (Thermo Scientific, #9990402) and a glass coverslip (Epredia, #BB02400550AC13MNZ0). Whole organoid imaging was performed on unsliced organoids following the mLSR-3D protocol with minor changes 77 . Briefly, blocking and permeabilization of whole fixed organoids was performed for 4 h at 4 °C in washing buffer 1 (0.2% Tween-20, 0.02% SDS, 0.2% Triton X-100 and 0.2% BSA in PBS).…”

Blueprints from plane to space: outlook of next‐generation three‐dimensional histopathology

“…Image quality was largely equivalent to conventionally acquired H&E images. (E) Multispectral large‐scale single‐cell resolution 3D (mLSR‐3D) imaging following FUnGI tissue clearing and “on‐the‐fly” linear unmixing, enabling up to eight fluorophores during a single acquisition 29 . The resulting 3D volume of the low‐grade glioma successfully facilitated detailed analysis, such as nuclei counting and volume estimation.…”

“… 26 Besides the compatibility with conventional H&E staining, attempts to obtain 3D images with label‐free, multiplex multicolor staining or mRNA labeling have been made (Figure 3E,F ), further broadening the accessible data and improving our understanding of molecular mechanisms driving cancer heterogeneity. 27 , 28 , 29 , 30 , 31 Larger cohort studies and clinical trials are still awaited to assess how 3D histopathology affects diagnostic accuracy in relation to the patients' prognosis.…”

Infrared color-sorting metasurfaces

“…In optical systems, color sorting is a fundamental process that partitions broadband photon energy into multiple subbands spectrally. This procedure find utility in various applications, including optical networks [1], spectroscopy [2,3], and microscopy [4]. Arrays of spectral filters with steep roll-offs are commonly employed for this purpose, often resulting in lengthy and bulky catadioptric optical trains [5,6].…”