Tissue imaging in 3D using visible light is limited and various clearing techniques were developed to increase imaging depth, but none provides universal solution for all tissues at all developmental stages. In this review, we focus on different tissue clearing methods for 3D imaging of heart and vasculature, based on chemical composition (solvent-based, simple immersion, hyperhydration, and hydrogel embedding techniques). We discuss in detail compatibility of various tissue clearing techniques with visualization methods: fluorescence preservation, immunohistochemistry, nuclear staining, and fluorescent dyes vascular perfusion. We also discuss myocardium visualization using autofluorescence, tissue shrinking, and expansion. Then we overview imaging methods used to study cardiovascular system and live imaging. We discuss heart and vessels segmentation methods and image analysis. The review covers the whole process of cardiovascular system 3D imaging, starting from tissue clearing and its compatibility with various visualization methods to the types of imaging methods and resulting image analysis.

Institute of Anatomy 1st Faculty of Medicine Charles University Prague Czech Republic

Institute of Physiology Czech Academy of Science Prague Czech Republic

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Aanhaanen W.T.J., Mommersteeg M.T.M., Norden J., Wakker V., Vries C.de G., Anderson R.H., Kispert A., Moorman A.F.M., Christoffels V.M. Developmental origin, growth, and three-dimensional architecture of the atrioventricular conduction Axis of the mouse heart. Circ. Res. 2010;107:728–736. PubMed

Akerberg A.A., Burns C.E., Burns C.G., Nguyen C. Deep learning enables automated volumetric assessments of cardiac function in zebrafish. Dis. Model. Mech. 2019;12:dmm040188. PubMed PMC

Alon S., Huynh G.H., Boyden E.S. Expansion microscopy: enabling single cell analysis in intact biological systems. FEBS J. 2019;286:1482–1494. PubMed PMC

Al-Surmi A., Wirza R., Mahmod R., Khalid F., Dimon M.Z. A new human heart vessel identification, segmentation and 3D reconstruction mechanism. J. Cardiothorac. Surg. 2014;9:161. PubMed PMC

Ariel P. A beginner’s guide to tissue clearing. Int. J. Biochem. Cell Biol. 2017;84:35–39. PubMed PMC

Azaripour A., Lagerweij T., Scharfbillig C., Jadczak A.E., Willershausen B., Van Noorden C.J.F. A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue. Prog. Histochem. Cytochem. 2016;51:9–23. PubMed

Baek K.I., Ding Y., Chang C.-C., Chang M., Sevag Packard R.R., Hsu J.J., Fei P., Hsiai T.K. Advanced microscopy to elucidate cardiovascular injury and regeneration: 4D light-sheet imaging. Prog. Biophys. Mol. Biol. 2018;138:105–115. PubMed PMC

Becker K., Jährling N., Saghafi S., Weiler R., Dodt H.-U. Chemical clearing and dehydration of GFP expressing mouse brains. PLoS One. 2012;7 doi: 10.1371/journal.pone.0033916. PubMed DOI PMC

Beis D., Bartman T., Jin S.-W., Scott I.C., D’Amico L.A., Ober E.A., Verkade H., Frantsve J., Field H.A., Wehman A.J.D. Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development. Development. 2005;132:4193–4204. PubMed

Belle M., Godefroy D., Couly G., Malone S.A., Collier F., Giacobini P., Chédotal A. Tridimensional visualization and analysis of early human development. Cell. 2017;169:161–173.e12. PubMed

Bensley J.G., De Matteo R., Harding R., Black M.J. Three-dimensional direct measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections. Sci. Rep. 2016;6:1–10. PubMed PMC

Bharadwaj K.N., Spitz C., Shekhar A., Yalcin H.C., Butcher J.T. Computational fluid dynamics of developing avian outflow tract heart valves. Ann. Biomed. Eng. 2012;40:2212–2227. PubMed PMC

Blinder P., Tsai P.S., Kaufhold J.P., Knutsen P.M., Suhl H., Kleinfeld D. The cortical angiome: an interconnected vascular network with noncolumnar patterns of blood flow. Nat. Neurosci. 2013;16:889–897. PubMed PMC

Bohuslavova R., Cerychova R., Papousek F., Olejnickova V., Bartos M., Görlach A., Kolar F., Sedmera D., Semenza G.L., Pavlinkova G. HIF-1α is required for development of the sympathetic nervous system. Proc. Natl. Acad. Sci. U S A. 2019;116:13414–13423. PubMed PMC

Boselli F., Vermot J.J.M. Live imaging and modeling for shear stress quantification in the embryonic zebrafish heart. Methods. 2016;94:129–134. PubMed

Bryson J.L., Coles M.C., Manley N.R. A method for labeling vasculature in embryonic mice. J. Vis. Exp. 2011:e3267. doi: 10.3791/3267. PubMed DOI PMC

Buffinton C.M., Faas D., Sedmera D. Stress and strain adaptation in load-dependent remodeling of the embryonic left ventricle. Biomech. Model. Mechanobiol. 2013;12:1037–1051. PubMed PMC

Butcher J.T., Sedmera D., Guldberg R.E., Markwald R.R. Quantitative volumetric analysis of cardiac morphogenesis assessed through micro-computed tomography. Dev. Dyn. 2007;236:802–809. PubMed

Cahalan M.D., Parker I., Wei S.H., Miller M.J. Two-photon tissue imaging: seeing the immune system in a fresh light. Nat. Rev. Immunol. 2002;2:872–880. PubMed PMC

Cahoon C.K., Yu Z., Wang Y., Guo F., Unruh J.R., Slaughter B.D., Hawley R.S. Superresolution expansion microscopy reveals the three-dimensional organization of the Drosophila synaptonemal complex. Proc. Natl. Acad. Sci. U S A. 2017;114:E6857–E6866. PubMed PMC

Carrillo M., Chuecos M., Gandhi K., Bednov A., Moore D.L., Maher J., Ventolini G., Ji G., Schlabritz-Loutsevitch N. Optical tissue clearing in combination with perfusion and immunofluorescence for placental vascular imaging. Medicine (Baltimore) 2018;97:e12392. PubMed PMC

Cavallero S., Shen H., Yi C., Lien C.-L., Kumar S.R., Sucov H.M. CXCL12 signaling is essential for maturation of the ventricular coronary endothelial plexus and establishment of functional coronary circulation. Dev. Cell. 2015;33:469–477. PubMed PMC

Cebasek V., Erzen I., Vyhnal A., Janacek J., Ribaric S., Kubinova L. The estimation error of skeletal muscle capillary supply is significantly reduced by 3D method. Microvasc. Res. 2010;79:40–46. PubMed

Chapman S.C., Lawson A., MacArthur W.C., Wiese R.J., Loechel R.H., Burgos-Trinidad M., Wakefield J.K., Ramabhadran R., Mauch T.J., Schoenwolf G.C. Ubiquitous GFP expression in transgenic chickens using a lentiviral vector. Development. 2005;132:935–940. PubMed

Chen F., Tillberg P.W., Boyden E.S. Expansion microscopy. Science. 2015;347:543–548. PubMed PMC

Chen F., Wassie A.T., Cote A.J., Sinha A., Alon S., Asano S., Daugharthy E.R., Chang J.-B., Marblestone A., Church G.M. Nanoscale imaging of RNA with expansion microscopy. Nat. Methods. 2016;13:679–684. PubMed PMC

Chen C., Qin C., Qiu H., Tarroni G., Duan J., Bai W., Rueckert D. Deep learning for cardiac image segmentation: a review. Front. Cardiovasc. Med. 2020;7:25. PubMed PMC

Choi W.J., Maga A.M., Kim E.S., Wang R.K. A feasibility study of OCT for anatomical and vascular phenotyping of mouse embryo. J. Biophotonics. 2020;13:e201960225. PubMed

Choquet C., Kelly R.G., Miquerol L. Nkx2-5 defines distinct scaffold and recruitment phases during formation of the murine cardiac Purkinje fiber network. Nat. Commun. 2020;11:5300. PubMed PMC

Chozinski T.J., Halpern A.R., Okawa H., Kim H.-J., Tremel G.J., Wong R.O., Vaughan J.C. Expansion microscopy with conventional antibodies and fluorescent proteins. Nat. Methods. 2016;13:485–488. PubMed PMC

Chung K., Wallace J., Kim S.-Y., Kalyanasundaram S., Andalman A.S., Davidson T.J., Mirzabekov J.J., Zalocusky K.A., Mattis J., Denisin A.K. Structural and molecular interrogation of intact biological systems. Nature. 2013;497:332–337. PubMed PMC

Cooper S., Bakal C. Accelerating live single-cell signalling studies. Trends Biotechnol. 2017;35:422–433. PubMed

Corliss B.A., Mathews C., Doty R., Rohde G., Peirce S.M. Methods to label, image, and analyze the complex structural architectures of microvascular networks. Microcirculation. 2019;26:e12520. PubMed PMC

Costantini I., Cicchi R., Cicchi R., Silvestri L., Vanzi F., Vanzi F., Pavone F.S., Pavone F.S. In-vivo and ex-vivo optical clearing methods for biological tissues: review. Biomed. Opt. Express. 2019;10:5251–5267. PubMed PMC

Creswell L.L., Wyers S.G., Pirolo J.S., Perman W.H., Vannier M.W., Pasque M.K. Mathematical modeling of the heart using magnetic resonance imaging. IEEE Trans. Med. Imaging. 1992;11:581–589. PubMed

D’Amico L., Scott I.C., Jungblut B., Stainier D.Y. A mutation in zebrafish hmgcr1b reveals a role for isoprenoids in vertebrate heart-tube formation. Curr. Biol. 2007;17:252–259. PubMed

Degenhardt Karl, Wright Alexander C., Debra H., Arun P., Epstein Jonathan A. Rapid 3D phenotyping of cardiovascular development in mouse embryos by micro-CT with iodine staining. Circ. Cardiovasc. Imaging. 2010;3:314–322. PubMed PMC

Di Giovanna A.P., Tibo A., Silvestri L., Müllenbroich M.C., Costantini I., Allegra Mascaro A.L., Sacconi L., Frasconi P., Pavone F.S. Whole-brain vasculature reconstruction at the single capillary level. Sci. Rep. 2018;8:12573. PubMed PMC

Ding Y., Ma J., Langenbacher A.D., Baek K.I., Lee J., Chang C.-C., Hsu J.J., Kulkarni R.P., Belperio J., Shi W. Multiscale light-sheet for rapid imaging of cardiopulmonary system. JCI Insight. 2018;3:e121396. PubMed PMC

Di Gregorio S., Fedele M., Pontone G., Corno A.F., Zunino P., Vergara C., Quarteroni A. A computational model applied to myocardial perfusion in the human heart: From large coronaries to microvasculature. Journal of Computational Physics. 2021;424:109836. doi: 10.1016/j.jcp.2020.109836. DOI

Ding Y., Gudapati V., Lin R., Fei Y., Song S., Chang C.-C., In K., Wang Z., Roustaei M., Kuang D. Saak transform-based machine learning for light-sheet imaging of cardiac trabeculation. IEEE Trans. Biomed. Eng. 2021;68:225–235. PubMed PMC

Ecabert O., Peters J., Walker M.J., Ivanc T., Lorenz C., von Berg J., Lessick J., Vembar M., Weese J. Segmentation of the heart and great vessels in CT images using a model-based adaptation framework. Med. Image Anal. 2011;15:863–876. PubMed

Epah J., Pálfi K., Dienst F.L., Malacarne P.F., Bremer R., Salamon M., Kumar S., Jo H., Schürmann C., Brandes R.P. 3D imaging and quantitative analysis of vascular networks: a comparison of ultramicroscopy and micro-computed tomography. Theranostics. 2018;8:2117–2133. PubMed PMC

Ermakova O., Orsini T., Gambadoro A., Chiani F., Tocchini-Valentini G.P. Three-dimensional microCT imaging of murine embryonic development from immediate post-implantation to organogenesis: application for phenotyping analysis of early embryonic lethality in mutant animals. Mamm. Genome. 2018;29:245–259. PubMed PMC

Ertürk A., Becker K., Jährling N., Mauch C.P., Hojer C.D., Egen J.G., Hellal F., Bradke F., Sheng M., Dodt H.-U. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat. Protoc. 2012;7:1983–1995. PubMed

Faulkner E.L., Thomas S.G., Neely R.K. An introduction to the methodology of expansion microscopy. Int. J. Biochem. Cell Biol. 2020;124:105764. PubMed

Fei P., Lee J., Packard R.R.S., Sereti K.-I., Xu H., Ma J., Ding Y., Kang H., Chen H., Sung K. Cardiac light-sheet fluorescent microscopy for multi-scale and rapid imaging of architecture and function. Sci. Rep. 2016;6:22489. PubMed PMC

Fonseca C.G., Backhaus M., Bluemke D.A., Britten R.D., Chung J.D., Cowan B.R., Dinov I.D., Finn J.P., Hunter P.J., Kadish A.H. The Cardiac Atlas Project—an imaging database for computational modeling and statistical atlases of the heart. Bioinformatics. 2011;27:2288–2295. PubMed PMC

Forouhar A.S., Liebling M., Hickerson A., Nasiraei-Moghaddam A., Tsai H.-J., Hove J.R., Fraser S.E., Dickinson M.E., Gharib M. The embryonic vertebrate heart tube is a dynamic suction pump. Science. 2006;312:751–753. PubMed

Foster D.S., Nguyen A.T., Chinta M., Salhotra A., Jones R.E., Mascharak S., Titan A.L., Ransom R.C., da Silva O.L., Foley E., Briger E., Longaker M.T. A Clearing Technique to Enhance Endogenous Fluorophores in Skin and. Soft Tissue. Sci Rep. 2019;9:15791. doi: 10.1038/s41598-019-50359-x. PubMed DOI PMC

Frangi A.F., Niessen W.J., Viergever M.A. Three-dimensional modeling for functional analysis of cardiac images: a review. IEEE Trans. Med. Imaging. 2001;20:2–25. PubMed

Freifeld L., Odstrcil I., Förster D., Ramirez A., Gagnon J.A., Randlett O., Costa E.K., Asano S., Celiker O.T., Gao R. Expansion microscopy of zebrafish for neuroscience and developmental biology studies. Proc. Natl. Acad. Sci. U S A. 2017;114:E10799–E10808. PubMed PMC

Gagnon L., Smith A.F., Boas D.A., Devor A., Secomb T.W., Sakadžić S. Modeling of cerebral oxygen transport based on in vivo microscopic imaging of microvascular network structure, blood flow, and oxygenation. Front. Comput. Neurosci. 2016;10:82. PubMed PMC

Gao M., Maraspini R., Beutel O., Zehtabian A., Eickholt B., Honigmann A., Ewers H. Expansion stimulated emission depletion microscopy (ExSTED) ACS Nano. 2018;12:4178–4185. PubMed

Gao R., Asano S.M., Boyden E.S. Q&A: expansion microscopy. BMC Biol. 2017;15:50. PubMed PMC

Ghanavati S., Lerch J.P., Sled J.G. Automatic anatomical labeling of the complete cerebral vasculature in mouse models. Neuroimage. 2014;95:117–128. PubMed

Gkontra P., Norton K.-A., Żak M.M., Clemente C., Agüero J., Ibáñez B., Santos A., Popel A.S., Arroyo A.G. Deciphering microvascular changes after myocardial infarction through 3D fully automated image analysis. Sci. Rep. 2018;8:1854. PubMed PMC

Gómez-Gaviro M.V., Sanderson D., Ripoll J., Desco M. Biomedical applications of tissue clearing and three-dimensional imaging in health and disease. iScience. 2020;23:101432. PubMed PMC

Goodyer W.R., Beyersdorf B.M., Paik D.T., Tian L., Li G., Buikema J.W., Chirikian O., Choi S., Venkatraman S., Adams E.L. Transcriptomic profiling of the developing cardiac conduction system at single-cell resolution. Circ. Res. 2019;125:379–397. PubMed PMC

Hahn A., Bode J., Alexander A., Karimian-Jazi K., Schregel K., Schwarz D., Sommerkamp A.C., Krüwel T., Abdollahi A., Wick W. Large-scale characterization of the microvascular geometry in development and disease by tissue clearing and quantitative ultramicroscopy. J. Cereb. Blood Flow Metab. 2020 271678X20961854. PubMed PMC

Halpern A.R., Alas G.C., Chozinski T.J., Paredez A.R., Vaughan J.C. Hybrid structured illumination expansion microscopy reveals microbial cytoskeleton organization. ACS Nano. 2017;11:12677–12686. PubMed PMC

Hama H., Hioki H., Namiki K., Hoshida T., Kurokawa H., Ishidate F., Kaneko T., Akagi T., Saito T., Saido T., Miyawaki A. ScaleS: an optical clearing palette for biological imaging. Nat. Neurosci. 2015;18:1518–1529. PubMed

Hasan M.R., Herz J., Hermann D.M., Doeppner T.R. Visualization of macroscopic cerebral vessel anatomy--a new and reliable technique in mice. J. Neurosci. Methods. 2012;204:249–253. PubMed

He L., Liu Q., Hu T., Huang X., Zhang H., Tian X., Yan Y., Wang L., Huang Y., Miquerol L. Genetic lineage tracing discloses arteriogenesis as the main mechanism for collateral growth in the mouse heart. Cardiovasc. Res. 2016;109:419–430. PubMed PMC

Honig M.G., Hume R.I. Dil and diO: versatile fluorescent dyes for neuronal labelling and pathway tracing. Trends Neurosci. 1989;12:340–341. PubMed

Hou B., Zhang D., Zhao S., Wei M., Yang Z., Wang S., Wang J., Zhang X., Liu B., Fan L. Scalable and DiI-compatible optical clearance of the mammalian brain. Front. Neuroanat. 2015;9:19. PubMed PMC

Hu N., Christensen D.A., Agrawal A.K., Beaumont C., Clark E.B., Hawkins J.A. Dependence of aortic arch morphogenesis on intracardiac blood flow in the left atrial ligated chick embryo. Anat. Rec. 2009;292:652–660. PubMed

Ivanovitch K., Temiño S., Torres M. Live imaging of heart tube development in mouse reveals alternating phases of cardiac differentiation and morphogenesis. Elife. 2017;6:e30668. PubMed PMC

Jensen B., Boukens B.J.D., Postma A.V., Gunst Q.D., van den Hoff M.J.B., Moorman A.F.M., Wang T., Christoffels V.M. Identifying the evolutionary building blocks of the cardiac conduction system. PLoS One. 2012;7:e44231. PubMed PMC

Ivins S., Roberts C., Vernay B., Scambler P.J. Analysis of coronary vessels in cleared embryonic hearts. J. Vis. Exp. 2016:54800. PubMed PMC

Jensen K.H.R., Berg R.W. CLARITY-compatible lipophilic dyes for electrode marking and neuronal tracing. Sci. Rep. 2016;6:32674. PubMed PMC

Jilani S.M., Murphy T.J., Thai S.N.M., Eichmann A., Alva J.A., Iruela-Arispe M.L. Selective binding of lectins to embryonic chicken vasculature. J. Histochem. Cytochem. 2003;51:597–604. PubMed

Junaid T.O., Bradley R.S., Lewis R.M., Aplin J.D., Johnstone E.D. Whole organ vascular casting and microCT examination of the human placental vascular tree reveals novel alterations associated with pregnancy disease. Sci. Rep. 2017;7:4144. PubMed PMC

Kapuscinski J. DAPI: a DNA-specific fluorescent probe. Biotech. Histochem. 1995;70:220–233. PubMed

Kattan J., Dettman R.W., Bristow J. Formation and remodeling of the coronary vascular bed in the embryonic avian heart. Dev. Dyn. 2004;230:34–43. PubMed

Ke M.-T., Fujimoto S., Imai T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat. Neurosci. 2013;16:1154–1161. PubMed

Keller P.J., Dodt H.-U. Light sheet microscopy of living or cleared specimens. Curr. Opin. Neurobiol. 2012;22:138–143. PubMed

Kennel P., Dichamp J., Barreau C., Guissard C., Teyssedre L., Rouquette J., Colombelli J., Lorsignol A., Casteilla L., Plouraboué F. From whole-organ imaging to in-silico blood flow modeling: a new multi-scale network analysis for revisiting tissue functional anatomy. PLoS Comput. Biol. 2020;16:e1007322. PubMed PMC

Kennel P., Teyssedre L., Colombelli J., Plouraboué F. Toward quantitative three-dimensional microvascular networks segmentation with multiview light-sheet fluorescence microscopy. JBO. 2018;23:086002. PubMed

Khoradmehr A., Mazaheri F., Anvari M., Tamadon A. A Simple Technique for Three-Dimensional Imaging and Segmentation of Brain Vasculature U sing Fast Free-of-Acrylamide Clearing Tissue in Murine. Cell J. 2019;21:49–56. doi: 10.22074/cellj.2019.5684. PubMed DOI PMC

Kidokoro H., Yonei-Tamura S., Tamura K., Schoenwolf G.C., Saijoh Y. The heart tube forms and elongates through dynamic cell rearrangement coordinated with foregut extension. Development. 2018;145:dev152488. PubMed PMC

Kim D.H., Ahn H.H., Sun W., Rhyu I.J. Electrophoretic tissue clearing and labeling methods for volume imaging of whole organs. AM. 2016;46:134–139.

Kim S.-Y., Cho J.H., Murray E., Bakh N., Choi H., Ohn K., Ruelas L., Hubbert A., McCue M., Vassallo S.L. Stochastic electrotransport selectively enhances the transport of highly electromobile molecules. Proc. Natl. Acad. Sci. U SA. 2015;112:E6274–E6283. PubMed PMC

Kirst C., Skriabine S., Vieites-Prado A., Topilko T., Bertin P., Gerschenfeld G., Verny F., Topilko P., Michalski N., Tessier-Lavigne M., Renier N. Mapping the fine-scale organization and plasticity of the brain vasculature. Cell. 2020;180:780–795.e25. PubMed

Klingberg A., Hasenberg A., Ludwig-Portugall I., Medyukhina A., Männ L., Brenzel A., Engel D.R., Figge M.T., Kurts C., Gunzer M. Fully automated evaluation of total glomerular number and capillary tuft size in nephritic kidneys using lightsheet microscopy. J. Am. Soc. Nephrol. 2017;28:452–459. PubMed PMC

Kolesová H., Bartoš M., Hsieh W.C., Olejníčková V., Sedmera D. Novel approaches to study coronary vasculature development in mice. Dev Dyn. 2018;247:1018–1027. doi: 10.1002/dvdy.24637. PubMed DOI

Kolesova H., Capek M., Radochova B., Janacek J., Sedmera D. Comparison of different tissue clearing methods and 3D imaging techniques for visualization of GFP-expressing mouse embryos and embryonic hearts. Histochem. Cell. Biol. 2016;146:141–152. PubMed

Kugler E., Plant K., Chico T., Armitage P. Enhancement and segmentation Workflow for the developing zebrafish vasculature. J. Imaging. 2019;5:14. PubMed PMC

Lagerweij T., Dusoswa S.A., Negrean A., Hendrikx E.M.L., de Vries H.E., Kole J., Garcia-Vallejo J.J., Mansvelder H.D., Vandertop W.P., Noske D.P. Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment. Angiogenesis. 2017;20:533–546. PubMed PMC

Lankford K.L., Arroyo E.J., Nazimek K., Bryniarski K., Askenase P.W., Kocsis J.D. Intravenously delivered mesenchymal stem cell-derived exosomes target M2-type macrophages in the injured spinal cord. PLoS One. 2018;13:e0190358. PubMed PMC

Lansford R., Rugonyi S. Follow me! A tale of avian heart development with comparisons to mammal heart development. J. Cardiovasc. Dev. Dis. 2020;7:8. PubMed PMC

Lapierre-Landry M., Kolesova H., Liu Y., Watanabe M., Jenkins M.W. Three-dimensional alignment of microvasculature and cardiomyocytes in the developing ventricle. Sci. Rep. 2020;10:14955. PubMed PMC

Lawson N.D., Weinstein B.M. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 2002;248:307–318. PubMed

Le N.A., Kuo W., Müller B., Kurtcuoglu V., Spingler B. Crosslinkable polymeric contrast agent for high-resolution X-ray imaging of the vascular system. Chem. Commun. (Camb.) 2020;56:5885–5888. PubMed

Lee E., Choi J., Jo Y., Kim J.Y., Jang Y.J., Lee H.M., Kim S.Y., Lee H.-J., Cho K., Jung N. ACT-PRESTO: rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging. Sci. Rep. 2016;6:18631. PubMed PMC

Lee H., Park J.-H., Seo I., Park S.-H., Kim S. Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine. BMC Dev. Biol. 2014;14:48. PubMed PMC

Lee S.-E., Nguyen C., Yoon J., Chang H.-J., Kim S., Kim C.H., Li D. Three-dimensional cardiomyocytes structure revealed by diffusion tensor imaging and its validation using a tissue-clearing technique. Sci. Rep. 2018;8:6640. PubMed PMC

Li G., Liu T., Tarokh A., Nie J., Guo L., Mara A., Holley S., Wong S.T. 3D cell nuclei segmentation based on gradient flow tracking. BMC Cell Biol. 2007;8:40. PubMed PMC

Li Y., Song Y., Zhao L., Gaidosh G., Laties A.M., Wen R. Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI. Nat. Protoc. 2008;3:1703–1708. PubMed PMC

Li Jingjing, Miao L., Shieh D., Spiotto E., Li J., Zhou B., Paul A., Schwartz R.J., Firulli A.B., Singer H.A. Single-cell lineage tracing reveals that oriented cell division contributes to trabecular morphogenesis and regional specification. Cell Rep. 2016;15:158–170. PubMed PMC

Liebling M., Forouhar A.S., Gharib M., Fraser S.E., Dickinson M.E. Four-dimensional cardiac imaging in living embryos via postacquisition synchronization of nongated slice sequences. J. Biomed. Opt. 2005;10:054001. PubMed

Liu Y., Broberg M.C.G., Watanabe M., Rollins A.M., Jenkins M.W. SLIME: robust, high-speed 3D microvascular mapping. Sci. Rep. 2019;9:893. PubMed PMC

Liu A.K.L., Lai H.M., Chang R.C.-C., Gentleman S.M. Free of acrylamide sodium dodecyl sulphate (SDS)-based tissue clearing (FASTClear): a novel protocol of tissue clearing for three-dimensional visualization of human brain tissues. Neuropathol Appl Neurobiol. 2017;43:346–351. doi: 10.1111/nan.12361. PubMed DOI PMC

Lowe K.L., Finney B.A., Deppermann C., Hägerling R., Gazit S.L., Frampton J., Buckley C., Camerer E., Nieswandt B., Kiefer F., Watson S.P. Podoplanin and CLEC-2 drive cerebrovascular patterning and integrity during development. Blood. 2015;125:3769–3777. PubMed PMC

Lugo-Hernandez E., Squire A., Hagemann N., Brenzel A., Sardari M., Schlechter J., Sanchez-Mendoza E.H., Gunzer M., Faissner A., Hermann D.M. 3D visualization and quantification of microvessels in the whole ischemic mouse brain using solvent-based clearing and light sheet microscopy. J. Cereb. Blood Flow Metab. 2017;37:3355–3367. PubMed PMC

Maeda K., Hata R., Hossmann K.A. Differences in the cerebrovascular anatomy of C57black/6 and SV129 mice. Neuroreport. 1998;9:1317–1319. PubMed

Matryba P., Kaczmarek L., Gołąb J. Advances in ex situ tissue optical clearing. Laser Photon. Rev. 2019;13:1800292.

Mellman K., Huisken J., Dinsmore C., Hoppe C., Stainier D.Y. Fibrillin-2b regulates endocardial morphogenesis in zebrafish. Dev. Biol. 2012;372:111–119. PubMed

Migliori B., Datta M.S., Dupre C., Apak M.C., Asano S., Gao R., Boyden E.S., Hermanson O., Yuste R., Tomer R. Light sheet theta microscopy for rapid high-resolution imaging of large biological samples. BMC Biol. 2018;16:57. PubMed PMC

Miller C.E., Thompson R.P., Bigelow M.R., Gittinger G., Trusk T.C., Sedmera D. Confocal imaging of the embryonic heart: how deep? Microscop. Microanal. 2005;11:216–223. PubMed

Miquerol L., Moreno-Rascon N., Beyer S., Dupays L., Meilhac S.M., Buckingham M.E., Franco D., Kelly R.G. Biphasic development of the mammalian ventricular conduction system. Circ. Res. 2010;107:153–161. PubMed

Mohan R.A., Mommersteeg M.T.M., Domínguez J.N., Choquet C., Wakker V., Vries C.de G., Boink G.J.J., Boukens B.J., Miquerol L., Verkerk A.O., Christoffels V.M. Embryonic Tbx3+ cardiomyocytes form the mature cardiac conduction system by progressive fate restriction. Development. 2018;145:dev167361. PubMed

Molbay M., Kolabas Z.I., Todorov M.I., Ohn T.-L., Ertürk A. A guidebook for DISCO tissue clearing. Mol Syst Biol. 2021;17:e9807. doi: 10.15252/msb.20209807. PubMed DOI PMC

Murakami T.C., Mano T., Saikawa S., Horiguchi S.A., Shigeta D., Baba K., Sekiya H., Shimizu Y., Tanaka K.F., Kiyonari H. A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing. Nat. Neurosci. 2018;21:625–637. PubMed

Murray E., Cho J.H., Goodwin D., Ku T., Swaney J., Kim S.-Y., Choi H., Park Y.-G., Park J.-Y., Hubbert A. Simple, scalable proteomic imaging for high-dimensional profiling of intact systems. Cell. 2015;163:1500–1514. PubMed PMC

Nagyova M., Slovinska L., Blasko J., Grulova I., Kuricova M., Cigankova V., Harvanova D., Cizkova D. A comparative study of PKH67, DiI, and BrdU labeling techniques for tracing rat mesenchymal stem cells. In Vitro Cell. Dev. Biol. Anim. 2014;50:656–663. PubMed

Nanka O., Krizova P., Fikrle M., Tuma M., Blaha M., Grim M., Sedmera D. Abnormal myocardial and coronary vasculature development in experimental hypoxia. Anat. Rec. (Hoboken) 2008;291:1187–1199. PubMed

Neckel P.H., Mattheus U., Hirt B., Just L., Mack A.F. Large-scale tissue clearing (PACT): Technical evaluation and new perspectives in immunofluorescence, histology, and ultrastructure. Sci Rep. 2016;6:34331. doi: 10.1038/srep34331. PubMed DOI PMC

Nehrhoff I., Bocancea D., Vaquero J., Vaquero J.J., Ripoll J., Desco M., Gómez-Gaviro M.V. 3D imaging in CUBIC-cleared mouse heart tissue: going deeper. Biomed. Opt. Express. 2016;7:3716–3720. PubMed PMC

Nguyen C.T., Lu Q., Wang Y., Chen J.-N. Zebrafish as a model for cardiovascular development and disease. Drug Discov. Today Dis. Models. 2008;5:135–140. PubMed PMC

Nojima S., Susaki E.A., Yoshida K., Takemoto H., Tsujimura N., Iijima S., Takachi K., Nakahara Y., Tahara S., Ohshima K. CUBIC pathology: three-dimensional imaging for pathological diagnosis. Sci. Rep. 2017;7:9269. PubMed PMC

Olejnickova V., Sankova B., Sedmera D., Janacek J. Trabecular architecture determines impulse propagation through the early embryonic mouse heart. Front. Physiol. 2018;9:1876. PubMed PMC

Packard R.R.S., Baek K.I., Beebe T., Jen N., Ding Yichen, Shi F., Fei P., Kang B.J., Chen P.-H., Gau J. Automated segmentation of light-sheet fluorescent imaging to characterize experimental doxorubicin-induced cardiac injury and repair. Sci. Rep. 2017;7:8603. PubMed PMC

Pan C., Cai R., Quacquarelli F.P., Ghasemigharagoz A., Lourbopoulos A., Matryba P., Plesnila N., Dichgans M., Hellal F., Ertürk A. Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat. Methods. 2016;13:859–867. PubMed

Park O.K., Kwak J., Jung Y.J., Kim Y.H., Hong H.-S., Hwang B.J., Kwon S.-H., Kee Y. 3D light-sheet fluorescence microscopy of cranial neurons and vasculature during zebrafish embryogenesis. Mol. Cells. 2015;38:975–981. PubMed PMC

Perbellini F., Liu A.K.L., Watson S.A., Bardi I., Rothery S.M., Terracciano C.M. Free-of-Acrylamide SDS-based Tissue Clearing (FASTClear) for three dimensional visualization of myocardial tissue. Sci. Rep. 2017;7:5188. PubMed PMC

Perdikaris P., Grinberg L., Karniadakis G.E. Multiscale modeling and simulation of brain blood flow. Phys. Fluids. 2016;28:021304. PubMed PMC

Plitman Mayo R. Advances in human placental biomechanics. Comput. Struct. Biotechnol. J. 2018;16:298–306. PubMed PMC

Proweller A., Wright A.C., Horng D., Cheng L., Lu M.M., Lepore J.J., Pear W.S., Parmacek M.S. Notch signaling in vascular smooth muscle cells is required to pattern the cerebral vasculature. Proc. Natl. Acad. Sci. U S A. 2007;104:16275–16280. PubMed PMC

Qi Y., Yu T., Xu J., Wan P., Ma Y., Zhu J., Li Y., Gong H., Luo Q., Zhu D. FDISCO: advanced solvent-based clearing method for imaging whole organs. Sci. Adv. 2019;5:eaau8355. PubMed PMC

Red-Horse K., Ueno H., Weissman I.L., Krasnow M.A. Coronary arteries form by developmental reprogramming of venous cells. Nature. 2010;464:549–553. PubMed PMC

Renier N., Adams E.L., Kirst C., Wu Z., Azevedo R., Kohl J., Autry A.E., Kadiri L., Umadevi Venkataraju K., Zhou Y. Mapping of brain activity by automated volume Analysis of immediate early genes. Cell. 2016;165:1789–1802. PubMed PMC

Richardson D.S., Lichtman J.W. Clarifying tissue clearing. Cell. 2015;162:246–257. PubMed PMC

Sandell L., Inman K., Trainor P. DAPI staining of whole-mount mouse embryos or fetal organs. Cold Spring Harb. Protoc. 2018;2018:prot094029. PubMed

Sarkar S., Schmued L. In vivo administration of fluorescent dextrans for the specific and sensitive localization of brain vascular pericytes and their characterization in normal and neurotoxin exposed brains. Neurotoxicology. 2012;33:436–443. PubMed

Sasse P., Malan D., Fleischmann M., Roell W., Gustafsson E., Bostani T., Fan Y., Kolbe T., Breitbach M., Addicks K. Perlecan is critical for heart stability. Cardiovasc. Res. 2008;80:435–444. PubMed

Schaad L., Hlushchuk R., Barré S., Gianni-Barrera R., Haberthür D., Banfi A., Djonov V. Correlative imaging of the murine hind limb vasculature and muscle tissue by MicroCT and light microscopy. Sci. Rep. 2017;7:41842. PubMed PMC

Schambach S.J., zag Bag S., Groden C., Schilling L., Brockmann M.A. Vascular imaging in small rodents using micro-CT. Methods. 2010;50:26–35. PubMed

Schürmann C., Gremse F., Jo H., Kiessling F., Brandes R.P. Micro-CT technique is well suited for documentation of remodeling processes in murine carotid arteries. PLoS One. 2015;10:e0130374. PubMed PMC

Schwarz M.K., Scherbarth A., Sprengel R., Engelhardt J., Theer P., Giese G. Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains. PLoS One. 2015;10:e0124650. PubMed PMC

Sedmera D., Misek I., Klima M., Thompson R.P. Heart development in the spotted dolphin (Stenella attenuata) Anat. Rec. A. Discov. Mol. Cell. Evol. Biol. 2003;273:687–699. PubMed

Sereti K.-I., Nguyen N.B., Kamran P., Zhao P., Ranjbarvaziri S., Park S., Sabri S., Engel J.L., Sung K., Kulkarni R.P. Analysis of cardiomyocyte clonal expansion during mouse heart development and injury. Nat. Commun. 2018;9:754. PubMed PMC

Shaikh Qureshi W.M., Miao L., Shieh D., Li J., Lu Y., Hu S., Barroso M., Mazurkiewicz J., Wu M. Imaging cleared embryonic and postnatal hearts at single-cell resolution. J. Vis. Exp. 2016;7:54303. PubMed PMC

Shan T., Zhao Y., Jiang S., Jiang H. In-vivo hemodynamic imaging of acute prenatal ethanol exposure in fetal brain by photoacoustic tomography. J. Biophotonics. 2020;13:e201960161. PubMed

Sharma B., Chang A., Red-Horse K. Coronary artery development: progenitor cells and differentiation pathways. Annu. Rev. Physiol. 2017;79:1–19. PubMed PMC

Sharpe J., Ahlgren U., Perry P., Hill B., Ross A., Hecksher-Sørensen J., Baldock R., Davidson D. Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science. 2002;296:541–545. PubMed

Silvestri L., Costantini I., Sacconi L., Pavone F.S. Clearing of fixed tissue: a review from a microscopist’s perspective. JBO. 2016;21:081205. PubMed

Sizarov A., Ya J., de Boer B.A., Lamers W.H., Christoffels V.M., Moorman A.F.M. formation of the building plan of the human heart: morphogenesis, growth, and differentiation. Circulation. 2011;123:1125–1135. PubMed

Stainier D., Fouquet B., Chen J.-N., Warren K.S., Weinstein B.M., Meiler S.E., Mohideen M., Neuhauss S., Solnica-Krezel L., Schier A.F. Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo. Development. 1996;123:285–292. PubMed

Stephens D.J., Allan V.J. Light microscopy techniques for live cell imaging. Science. 2003;300:82–86. PubMed

Sung K., Ding Y., Ma J., Chen H., Huang V., Cheng M., Yang C.F., Kim J.T., Eguchi D., Di Carlo D. Simplified three-dimensional tissue clearing and incorporation of colorimetric phenotyping. Sci. Rep. 2016;6:30736. PubMed PMC

Susaki E.A., Tainaka K., Perrin D., Kishino F., Tawara T., Watanabe T.M., Yokoyama C., Onoe H., Eguchi M., Yamaguchi S. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell. 2014;157:726–739. PubMed

Susaki E.A., Tainaka K., Perrin D., Yukinaga H., Kuno A., Ueda H.R. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc. 2015;10:1709–1727. doi: 10.1038/nprot.2015.085. PubMed DOI

Tainaka K., Kubota S.I., Suyama T.Q., Susaki E.A., Perrin D., Ukai-Tadenuma M., Ukai H., Ueda H.R. Whole-body imaging with single-cell resolution by tissue decolorization. Cell. 2014;159:911–924. PubMed

Tainaka K., Kuno A., Kubota S.I., Murakami T., Ueda H.R. Chemical principles in tissue clearing and staining protocols for whole-body cell profiling. Annu. Rev. Cell Dev. Biol. 2016;32:713–741. PubMed

Tarnowski B.I., Spinale F.G., Nicholson J.H. DAPI as a useful stain for nuclear quantitation. Biotech. Histochem. 1991;66:297–302. PubMed

Taylor J.M., Nelson C.J., Bruton F.A., Baghbadrani A.K., Buckley C., Tucker C.S., Rossi A.G., Mullins J.J., Denvir M.A. Adaptive prospective optical gating enables day-long 3D time-lapse imaging of the beating embryonic zebrafish heart. Nat. Commun. 2019;10:1–15. PubMed PMC

Tian T., Yang Z., Li X. Tissue clearing technique: recent progress and biomedical applications. J. Anat. 2021;238:489–507. PubMed PMC

Tillberg P.W., Chen F., Piatkevich K.D., Zhao Y., Yu C.-C., Jay), English B.P., Gao L., Martorell A., Suk H.-J. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat. Biotechnol. 2016;34:987–992. PubMed PMC

Todorov M.I., Paetzold J.C., Schoppe O., Tetteh G., Shit S., Efremov V., Todorov-Völgyi K., Düring M., Dichgans M., Piraud M. Machine learning analysis of whole mouse brain vasculature. Nat. Methods. 2020;17:442–449. PubMed PMC

Tomer R., Ye L., Hsueh B., Deisseroth K. Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat. Protoc. 2014;9:1682–1697. PubMed PMC

Traver D., Paw B.H., Poss K.D., Penberthy W.T., Lin S., Zon L.I. Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nat. Immunol. 2003;4:1238–1246. PubMed

Treweek J.B., Chan K.Y., Flytzanis N.C., Yang B., Deverman B.E., Greenbaum A., Lignell A., Xiao C., Cai L., Ladinsky M.S. Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. Nat. Protoc. 2015;10:1860–1896. PubMed PMC

Truong T.V., Supatto W., Koos D.S., Choi J.M., Fraser S.E. Deep and fast live imaging with two-photon scanned light-sheet microscopy. Nat. Methods. 2011;8:757–760. PubMed

Tsai P.S., Kaufhold J.P., Blinder P., Friedman B., Drew P.J., Karten H.J., Lyden P.D., Kleinfeld D. Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocalization of nuclei and vessels. J Neurosci. 2009;29:14553–14570. doi: 10.1523/JNEUROSCI.3287-09.2009. PubMed DOI PMC

Ueda H.R., Ertürk A., Chung K., Gradinaru V., Chédotal A., Tomancak P., Keller P.J. Tissue clearing and its applications in neuroscience. Nat. Rev. Neurosci. 2020;21:61–79. PubMed PMC

van Eif V.W.W., Devalla H.D., Boink G.J.J., Christoffels V.M. Transcriptional regulation of the cardiac conduction system. Nat. Rev. Cardiol. 2018;15:617–630. PubMed

van Zandvoort M., Engels W., Douma K., Beckers L., Oude Egbrink M., Daemen M., Slaaf D.W. Two-photon microscopy for imaging of the (atherosclerotic) vascular wall: a proof of concept study. J. Vasc. Res. 2004;41:54–63. PubMed

Veith A., B Baker A. A rapid, nondestructive method for vascular network visualization. Biotechniques. 2020;69:443–449. PubMed PMC

Vigouroux R.J., Belle M., Chédotal A. Neuroscience in the third dimension: shedding new light on the brain with tissue clearing. Mol. Brain. 2017;10:33. PubMed PMC

Vrbacky M., Kovalcikova J., Chawengsaksophak K., Beck I.M., Mracek T., Nuskova H., Sedmera D., Papousek F., Kolar F., Sobol M. Knockout of Tmem70 alters biogenesis of ATP synthase and leads to embryonal lethality in mice. Hum. Mol. Genet. 2016;25:4674–4685. PubMed

Walter T., Shattuck D.W., Baldock R., Bastin M.E., Carpenter A.E., Duce S., Ellenberg J., Fraser A., Hamilton N., Pieper S. Visualization of image data from cells to organisms. Nat. Methods. 2010;7:S26–S41. PubMed PMC

Wang D., Wang Y., Wang W., Luo D., Chitgupi U., Geng J., Zhou Y., Wang L., Lovell J.F., Xia J. Deep tissue photoacoustic computed tomography with a fast and compact laser system. Biomed. Opt. Express. 2016;8:112–123. PubMed PMC

Wang Y., Dur O., Patrick M.J., Tinney J.P., Tobita K., Keller B.B., Pekkan K. Aortic arch morphogenesis and flow modeling in the chick embryo. Ann. Biomed. Eng. 2009;37:1069–1081. PubMed

Wang Z., Zhang Jie, Fan G., Zhao H., Wang X., Zhang Jing, Zhang P., Wang W. Imaging transparent intact cardiac tissue with single-cell resolution. Biomed. Opt. Express. 2018;9:423. PubMed PMC

Wassie A.T., Zhao Y., Boyden E.S. Expansion microscopy: principles and uses in biological research. Nat. Methods. 2019;16:33–41. PubMed PMC

Weninger W.J., Geyer S.H., Mohun T.J., Rasskin-Gutman D., Matsui T., Ribeiro I., Costa L.da F., Izpisúa-Belmonte J.C., Müller G.B. High-resolution episcopic microscopy: a rapid technique for high detailed 3D analysis of gene activity in the context of tissue architecture and morphology. Anat. Embryol. 2006;211:213–221. PubMed

Wu Z., Rademakers T., Kiessling F., Vogt M., Westein E., Weber C., Megens R.T.A., van Zandvoort M. Multi-photon microscopy in cardiovascular research. Methods. 2017;130:79–89. PubMed

Xian Z., Wang X., Yan S., Yang D., Chen J., Peng C. Main coronary vessel segmentation using deep learning in smart medical. Math. Probl. Eng. 2020:8858344. doi: 10.1155/2020/8858344. DOI

Xu H., Tong Z., Ye Q., Sun T., Hong Z., Zhang L., Bortnick A., Cho S., Beuzer P., Axelrod J. Molecular organization of mammalian meiotic chromosome axis revealed by expansion STORM microscopy. Proc. Natl. Acad. Sci. U S A. 2019;116:18423–18428. PubMed PMC

Xu J., Ma Y., Yu T., Zhu D. Quantitative assessment of optical clearing methods in various intact mouse organs. J. Biophotonics. 2019;12:e201800134. doi: 10.1002/jbio.201800134. PubMed DOI

Yalcin H.C., Shekhar A., McQuinn T.C., Butcher J.T. Hemodynamic patterning of the avian atrioventricular valve. Dev. Dyn. 2011;240:23–35. PubMed PMC

Yang S., Kweon J., Roh J.-H., Lee J.-H., Kang H., Park L.-J., Kim D.J., Yang H., Hur J., Kang D.-Y. Deep learning segmentation of major vessels in X-ray coronary angiography. Sci. Rep. 2019;9:16897. PubMed PMC

Yang B., Treweek J.B., Kulkarni R.P., Deverman B.E., Chen C.-K., Lubeck E., Shah S., Cai L., Gradinaru V. Single-Cell Phenotyping within Transparent Intact Tissue Through Whole-Body Clearing. Cell. 2014;158:945–958. doi: 10.1016/j.cell.2014.07.017. PubMed DOI PMC

Yokoyama T., Lee J.-K., Miwa K., Opthof T., Tomoyama S., Nakanishi H., Yoshida A., Yasui H., Iida T., Miyagawa S. Quantification of sympathetic hyperinnervation and denervation after myocardial infarction by three-dimensional assessment of the cardiac sympathetic network in cleared transparent murine hearts. PLoS One. 2017;12:e0182072. PubMed PMC

Yue Y., Zong W., Li X., Li J., Zhang Y., Wu R., Liu Y., Cui J., Wang Q., Bian Y. Long-term, in toto live imaging of cardiomyocyte behaviour during mouse ventricle chamber formation at single-cell resolution. PLoS One. 2020;15:e0226791. PubMed

Zagorchev L., Oses P., Zhuang Z.W., Moodie K., Mulligan-Kehoe M.J., Simons M., Couffinhal T. Micro computed tomography for vascular exploration. J. Angiogenes. Res. 2010;2:7. PubMed PMC

Zhang H., Chalothorn D., Faber J.E. Collateral vessels have unique endothelial and smooth muscle cell phenotypes. Int. J. Mol. Sci. 2019;20:3608. PubMed PMC

Zhang Z.G., Zhang L., Tsang W., Soltanian-Zadeh H., Morris D., Zhang R., Goussev A., Powers C., Yeich T., Chopp M. Correlation of VEGF and angiopoietin expression with disruption of blood-brain barrier and angiogenesis after focal cerebral ischemia. J. Cereb. Blood Flow Metab. 2002;22:379–392. PubMed

Zhao X., Wu J., Gray C.D., McGregor K., Rossi A.G., Morrison H., Jansen M.A., Gray G.A. Optical projection tomography permits efficient assessment of infarct volume in the murine heart postmyocardial infarction. Am. J. Physiol. Heart Circ. Physiol. 2015;309:H702–H710. PubMed PMC

Zhou B., Ma Q., Kong S.W., Hu Y., Campbell P.H., McGowan F.X., Ackerman K.G., Wu B., Zhou B., Tevosian S.G., Pu W.T. Fog2 is critical for cardiac function and maintenance of coronary vasculature in the adult mouse heart. J. Clin. Invest. 2009;119:1462–1476. PubMed PMC

Zhu J., Yu T., Li Y., Xu J., Qi Y., Yao Y., Ma Y., Wan P., Chen Z., Li X. MACS: rapid aqueous clearing system for 3D mapping of intact organs. Adv. Sci. (Weinh) 2020;7:1903185. PubMed PMC

Zucker, R.M., Hunter, S., Rogers, J.M., 1998. Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos. Cytometry 33, 348–354. 10.1002/(sici)1097-0320(19981101)33:3%3C348::aid-cyto9%3E3.0.co;2-c. PubMed DOI