EyeCi: Optical clearing and imaging of immunolabeled mouse eyes using light-sheet fluorescence microscopy

Henning, Yoshiyuki; Osadnik, Christin; Malkemper, E. Pascal

doi:10.1016/j.exer.2018.12.001

Cited by 46 publications

(47 citation statements)

References 29 publications

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“…During the preparation of our manuscript, a pioneering study reported a tissue clearing method for the eye that combined melanin bleaching and the iDISCO tissue clearing method. 34 Although this report visualized the distribution patterns of vessels and S-opsin, it was unclear whether tissue clearing methods could be applied to various kinds of cells and structures in the retina. In addition, it was also unclear whether tissue clearing methods could be applicable to larger eyes from other animals.…”

Section: Discussionmentioning

confidence: 95%

Visualization of the Retina in Intact Eyes of Mice and Ferrets Using a Tissue Clearing Method

Duong

Saito

et al. 2020

Trans. Vis. Sci. Tech.

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Purpose Visualization of specific cells and structures in intact organs would greatly facilitate our knowledge about pathological changes; therefore, a tissue clearing method applicable to the intact eye may be valuable. Here we report a novel imaging method for the retina using the hyperhydration-based tissue clearing technique CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational Analysis). Methods Eyes of Institute of Cancer Research (ICR) mice, C57BL/6 mice, and normally pigmented sable ferrets ( Mustela putorius furo ) were used. Intact eyes were subjected to CUBIC, melanin bleaching with H 2 O 2 , and immunostaining. Images of the retina in intact eyes were taken using epifluorescence microscopes and confocal microscopes. Results The combination of melanin bleaching and CUBIC efficiently made the eyes of C57BL/6 mice transparent. By combining melanin bleaching, CUBIC, and immunostaining, we succeeded in visualization of retinal structures from the outside of the intact eyes of mice. Furthermore, we found that our methods were applicable not only to mouse eyes but also to ferret eyes, which are much larger than those of mice. Conclusions Our method was useful for visualizing specific cells and structures in the retina of intact eyes with single-cell resolution without making tissue sections. Translational Relevance This simple and efficient method can be applicable to various rodent models, including those associated with glaucoma or myopia, and will facilitate evaluating the effects of novel therapy for relevant eye diseases by visualizing changes from the retina to the sclera at both molecular and macroscopic levels simultaneously in a whole-eye preparation.

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Section: Discussionmentioning

confidence: 95%

Visualization of the Retina in Intact Eyes of Mice and Ferrets Using a Tissue Clearing Method

Duong

Saito

et al. 2020

Trans. Vis. Sci. Tech.

View full text Add to dashboard Cite

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“…iDISCO is an optical clearing method that makes biological samples more transparent ("cleared") ( Fig. 2) and has been successfully used to image three-dimensional structures, including intact mouse organs such as the brain, kidney, intestine, eye, and even whole embryos [18,[22][23][24][25]. Recently, Becher et al [26] applied the iDIS-CO technology for 3D profiling of atherosclerotic plaques and arterial remodeling after carotid artery ligation.…”

Section: Resultsmentioning

confidence: 99%

Three-Dimensional Imaging of Intraplaque Neovascularization in a Mouse Model of Advanced Atherosclerosis

et al. 2020

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Multiple lines of evidence suggest that intraplaque (IP) neovascularization promotes atherosclerotic plaque growth, destabilization, and rupture. However, pharmacological inhibition of IP neovascularization remains largely unexplored due to the limited number of animal models that develop IP neovessels and the lack of reliable methods for visualizing IP angiogenesis. Here, we applied 3D confocal microscopy with an optimized tissue-clearing process, immunolabeling-enabled three-dimensional imaging of solvent-cleared organs, to visualize IP neovessels in apolipoprotein E-deficient (ApoE −/−) mice carrying a heterozygous mutation (C1039+/−) in the fibrillin-1 gene. Unlike regular ApoE −/− mice, this mouse model is characterized by the presence of advanced plaques with evident IP neovascularization. Plaques were stained with antibodies against endothelial marker CD31 for 3 days, followed by incubation with fluorescently labeled secondary antibodies. Subsequent tissue clearing with dichloromethane (DCM)/methanol, DCM, and dibenzyl ether allowed easy visualization and 3D reconstruction of the IP vascular network while plaque morphology remained intact.

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“…Ovarian tissue and murine ovarian follicles have been cleared with CUBIC in EGFP transgenic mice ( Kagami et al., 2018 ). In the study of the eye, an intact mouse eye has been cleared with a new protocol called EyeCi, bleaching the melanin with hydrogen peroxide and using solvent-based clearing, which enables ocular and retinal vascular and neovascularization ( Henning et al., 2019 ). CUBIC has also been combined with hydrogen peroxide to clear the mouse eye.…”

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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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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EyeCi: Optical clearing and imaging of immunolabeled mouse eyes using light-sheet fluorescence microscopy

Cited by 46 publications

References 29 publications

Visualization of the Retina in Intact Eyes of Mice and Ferrets Using a Tissue Clearing Method

Visualization of the Retina in Intact Eyes of Mice and Ferrets Using a Tissue Clearing Method

Three-Dimensional Imaging of Intraplaque Neovascularization in a Mouse Model of Advanced Atherosclerosis

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

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