MACS: Rapid Aqueous Clearing System for 3D Mapping of Intact Organs

Zhu, Jingtan; Yu, Tingting; Li, Yusha; Xu, Jianyi; Qi, Yisong; Yao, Yingtao; Ma, Yilin; Wan, Peng; Chen, Zhilong; Li, Xiangning; Gong, Hui; Luo, Qingming; Zhu, Dan

doi:10.1002/advs.201903185

Cited by 68 publications

(66 citation statements)

References 63 publications

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“… N ( Schwarz et al., 2015 ) Fly-clear Protease, acetone, Solution-1 (THEED, Triton X- and Urea), Solution-2 (meglumine diazotrate) A/O Drosophila melanogaster N Medium (1 w) N.D. Y ( Pende et al., 2018 ) FRUIT Fructose and urea A Rabbit brain No shrinkage for 30% FRUIT Short (4 d) Y Y ( Hou et al., 2015 ) iDISCO+ Methanol, dichloromethane, dibenzyl ether O Mouse and rat brains N Very long (18 d) Y N.D. ( Renier et al., 2014 ) ( Perin et al., 2019 ) IsoScaleSQ 9.1 M urea, 22.5% (v/w) d-sorbitol, 200 mM sodium chloride, and 2% (v/w) Triton X-100. A Mouse brain N Very short (30 min) Y (thin slices) N ( Sato et al., 2019 ) LUCID TDE, glycerol, TBS, Tween 20 A Porcine gastrointestinal mucosa N Short (5–7 d) Y N.D. ( Hildebrand et al, 2018 ) MACS MXDA, sorbitol A Murine brain, heart, lung, spleen, femur, and whole body Slight expansion Fast (2.5d) Y N ( Zhu et al., 2020 ) MASH DISCO with RIMS substituted by trans-cinnamaldehyde, TDE, and wintergreen oil O Human cortex ND Medium Y ? ( Hildebrand et al, 2018 ) MYOCLEAR Hydrogel (VA-044, A4P0), PTwH A Mice diaphragm muscle...…”

Section: Transparency and Tissue Clearing Methodsmentioning

confidence: 99%

“…MXDA-based Aqueous Clearing System (MACS) is a clearing method that has been very recently developed, and that also reduces delipidation, allowing, therefore, lipid staining with Dil labeling or other markers, and that, nevertheless, maintains a high clearing performance. It uses m-xylylenediamine (MXDA), which is a water-miscible liquid with a high refractive Index that induces hyperhydration in a similar way to urea ( Zhu et al., 2020 ).…”

Section: Translational Applications Of Tissue Clearing and 3d Imagingmentioning

confidence: 99%

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Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

et al. 2020

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Summary Three-dimensional (3D) optical imaging techniques can expand our knowledge about physiological and pathological processes that cannot be fully understood with 2D approaches. Standard diagnostic tests frequently are not sufficient to unequivocally determine the presence of a pathological condition. Whole-organ optical imaging requires tissue transparency, which can be achieved by using tissue clearing procedures enabling deeper image acquisition and therefore making possible the analysis of large-scale biological tissue samples. Here, we review currently available clearing agents, methods, and their application in imaging of physiological or pathological conditions in different animal and human organs. We also compare different optical tissue clearing methods discussing their advantages and disadvantages and review the use of different 3D imaging techniques for the visualization and image acquisition of cleared tissues. The use of optical tissue clearing resources for large-scale biological tissues 3D imaging paves the way for future applications in translational and clinical research.

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Section: Transparency and Tissue Clearing Methodsmentioning

confidence: 99%

Section: Translational Applications Of Tissue Clearing and 3d Imagingmentioning

confidence: 99%

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

et al. 2020

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“…Like SeeDB2, Ce3D is also an iohexol-based medium, with the addition a of a small percentage of N-methylacetamide (Li et al, 2017). A recently reported novel medium, MACS (Zhu et al, 2020) is based on m-xylylenediamine, an interesting material, with a refractive index tunable up to 1.57. This appears to offer promise of good tissue clarity without lipid extraction and short processing times, although at the expense of m-xylylenediamine being highly toxic, which we note is a specific advantage of SeeDB2 and Rapiclear.…”

Section: Alternative Clearing Methods and Future Workmentioning

confidence: 99%

Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index

et al. 2020

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Improvement of imaging quality has the potential to visualize previously unseen building blocks of the brain and is therefore one of the great challenges in neuroscience. Rapid development of new tissue clearing techniques in recent years have attempted to solve imaging compromises in thick brain samples, particularly for high resolution optical microscopy, where the clearing medium needs to match the high refractive index of the objective immersion medium. These problems are exacerbated in insect tissue, where numerous (initially air-filled) tracheal tubes branching throughout the brain increase the scattering of light. To date, surprisingly few studies have systematically quantified the benefits of such clearing methods using objective transparency and tissue shrinkage measurements. In this study we compare a traditional and widely used insect clearing medium, methyl salicylate combined with permanent mounting in Permount (“MS/P”) with several more recently applied clearing media that offer tunable refractive index (n): 2,2′-thiodiethanol (TDE), “SeeDB2” (in variants SeeDB2S and SeeDB2G matched to oil and glycerol immersion, n = 1.52 and 1.47, respectively) and Rapiclear (also with n = 1.52 and 1.47). We measured transparency and tissue shrinkage by comparing freshly dissected brains with cleared brains from dipteran flies, with or without addition of vacuum or ethanol pre-treatments (dehydration and rehydration) to evacuate air from the tracheal system. The results show that ethanol pre-treatment is very effective for improving transparency, regardless of the subsequent clearing medium, while vacuum treatment offers little measurable benefit. Ethanol pre-treated SeeDB2G and Rapiclear brains show much less shrinkage than using the traditional MS/P method. Furthermore, at lower refractive index, closer to that of glycerol immersion, these recently developed media offer outstanding transparency compared to TDE and MS/P. Rapiclear protocols were less laborious compared to SeeDB2, but both offer sufficient transparency and refractive index tunability to permit super-resolution imaging of local volumes in whole mount brains from large insects, and even light-sheet microscopy. Although long-term permanency of Rapiclear stored samples remains to be established, our samples still showed good preservation of fluorescence after storage for more than a year at room temperature.

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“…The best protocol can differ depending on the research target or means of observation. Compatibility with DiIs was also demonstrated for a recently developed tissue clearing protocol, MACS 42 . This protocol also appears promising for clearing tissues after liposome-mediated vessel painting.…”

Section: Application Of Vessel Painting For Various Tissues Liposomementioning

confidence: 89%

Pathological application of carbocyanine dye-based multicolour imaging of vasculature and associated structures

Konno

Matsumoto

Tomono

et al. 2020

Sci Rep

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Simultaneous visualisation of vasculature and surrounding tissue structures is essential for a better understanding of vascular pathologies. in this work, we describe a histochemical strategy for threedimensional, multicolour imaging of vasculature and associated structures, using a carbocyanine dye-based technique, vessel painting. We developed a series of applications to allow the combination of vessel painting with other histochemical methods, including immunostaining and tissue clearing for confocal and two-photon microscopies. We also introduced a two-photon microscopy setup that incorporates an aberration correction system to correct aberrations caused by the mismatch of refractive indices between samples and immersion mediums, for higher-quality images of intact tissue structures. Finally, we demonstrate the practical utility of our approach by visualising fine pathological alterations to the renal glomeruli of igA nephropathy model mice in unprecedented detail. the technical advancements should enhance the versatility of vessel painting, offering rapid and costeffective methods for vascular pathologies. Blood vessels form a network that delivers molecules and cells throughout the body. Because of their threedimensional (3D) nature, it is difficult, or at least painstaking, to understand their distribution by observing conventional histological sections. Recent advancements in optical sectioning microscopy, such as confocal, multi-photon, and light-sheet microscopies, have made volume imaging of vasculature much easier. These fluorescence-based microscopies require effective and specific labelling techniques for imaging vasculature in 3D specimens. These techniques include genetically encoded fluorescent proteins, specific probes such as antibodies and lectins, or infusion of space-occupying materials 1-4. Vessel painting is a method of labelling the entire vasculature of small mammals through perfusion of a lipophilic carbocyanine dye, DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine) 5-9. Recently, we improved labelling intensity and uniformity, as well as the reproducibility of the technique, by introducing a neutral liposome and a hydrophilic DiI analogue 10. Although vessel painting is an easy and cost-effective technique to label vasculature intensely, it has several limitations. First, the excitation/emission spectra of DiI largely overlap with popular red fluorophores, such as Alexa 568 and mCherry, which limits the combination of probes for multi-labelling. Second, liposome-mediated vessel painting has only been applied to the vasculature of the central nervous system (CNS) of small rodents and its applicability to other organs needs to be tested 5-9. Third, its compatibility with various tissue clearing protocols has not been tested, with the exception of an expensive proprietary reagent of unknown composition, FocusClear 7. Finally, its compatibility for multi-labelling with other types of probes, such as antibodies or fluorescently labelled small molecules, except for nuclear stainin...

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MACS: Rapid Aqueous Clearing System for 3D Mapping of Intact Organs

Cited by 68 publications

References 63 publications

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index

Pathological application of carbocyanine dye-based multicolour imaging of vasculature and associated structures

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