Recent tissue-clearing approaches have become important alternatives to standard histology approaches. However, light scattering in thick tissues and the size restrictions on samples that can be imaged with standard light-sheet microscopy pose limitations for analyzing large samples such as an entire rodent body. We developed 'ultimate DISCO' (uDISCO) clearing to overcome these limitations in volumetric imaging. uDISCO preserves fluorescent proteins over months and renders intact organs and rodent bodies transparent while reducing their size up to 65%. We used uDISCO to image neuronal connections and vasculature from head to toe over 7 cm and to perform unbiased screening of transplanted stem cells within the entire body of adult mice. uDISCO is compatible with diverse labeling methods and archival human tissue, and it can readily be used in various biomedical applications to study organization of large organ systems throughout entire organisms.
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This work was supported by the Vascular Dementia Research Foundation, Synergy Excellence Cluster Munich (SyNergy), ERA-Net Neuron (01EW1501A to A.E. and N.P.), and the European Union's Horizon 2020 research and innovation programme (grant agreement no. 666881, SVDs@target, M.D.). A.L. and N.P. were supported by a Marie Curie Intra European Fellowship grant (FP7-PEOPLE-2013-IEF, project no. 625970). We thank M. Hübener and F. Voss (Max Planck Institute of Neurobiology, Munich) for providing mice; D. Trauner and O. Thorn-Seshold for helpful discussions; A. Weingart for illustrations; and C. Hojer, S. Tappan and T. Misgeld for critical reading of the manuscript. C.P. and R.C. are members of the Graduate School of Systemic Neurosciences (GSN), Ludwig Maximilian University of Munich. Human tissues were provided by the brain bank of the Institute of Anatomy, University of Leipzig.
A ~3 months old CX3CR1-EGFP mouse brain was cleared with uDISCO and imaged with the light-sheet microscopy using the 4x corrected objective. The individual microglia throughout the entire brain are evident.
Surface reconstruction and 3D maximum intensity projection of a paw with forearm from 4 months old C57BL/6N mouse labeled with Texas Red Dextran after whole-body uDISCO clearing. uDISCO renders the bones fully transparent, allowing the visualization of the vascular details in the entire limbs.
3D visualization and orthoslice of the forearm with vasculature labeling in Supplementary Video 2. Fine details of the vasculature are clear throughout the entire scan.
The vasculature of a 4-weeks old rat was labeled with Texas Red Dextran. We imaged the entire CNS (the brain plus spinal cord) after whole-body clearing of the rat. Both the large and small vessels are clearly visible throughout the entire scan of ≥13 cm CNS rat tissue.
3D visualization of neuronal connections throughout the intact mouse CNS in Figure 4 (GFP-M mouse, 4 months old). The fine details of the neuronal connections are evident from head to the nerves invading the hind limbs.
A single traced axons (red) in the spinal cord of the sample from Figure 4 is shown. The trajectories of individual axons in the entire CNS of the adult mice can be determined. Note that occasional curling of long spinal cord axons does not interfere with the tracing of their entire trajectories. The pseudo-colored traced axon is shown thicker than its actual size for easier visualization.
A GFP-M mouse brain (4 months old) was cleared with uDISCO and imaged with the light-sheet microscopy. The whole-brain images were obtained using the 4x corrected objective, and high-magnification images in the second part of the video were obtained using the 20x corrected objective.
A light-sheet microscopy stack acquired on uDISCO cleared GFP-M mouse brain at 0.5 – 1 mm depth using Zeiss CLARITY objective (25x, NA 1.0), which was directly immersed into BABB-D. The individual dendritic spines are evident throughout the scan.
The video shows the 3D reconstruction of the densely distributed BMSCs throughout the entire lung. uDISCO rendered the intact lungs fully transparent, allowing to assess the distribution of individual BMSCs.
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