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      Tissue clearing and imaging methods for cardiovascular development

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          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.

          Abstract

          Imaging Methods in Chemistry; Optical Imaging; Biology Experimental Methods

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          Single-cell phenotyping within transparent intact tissue through whole-body clearing.

          Bin Yang, Jennifer Treweek, Rajan Kulkarni … (2014)
          Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies. Copyright © 2014 Elsevier Inc. All rights reserved.
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            Optical imaging. Expansion microscopy.

            Fei Chen, Paul W. Tillberg, Edward S. Boyden (2015)
            In optical microscopy, fine structural details are resolved by using refraction to magnify images of a specimen. We discovered that by synthesizing a swellable polymer network within a specimen, it can be physically expanded, resulting in physical magnification. By covalently anchoring specific labels located within the specimen directly to the polymer network, labels spaced closer than the optical diffraction limit can be isotropically separated and optically resolved, a process we call expansion microscopy (ExM). Thus, this process can be used to perform scalable superresolution microscopy with diffraction-limited microscopes. We demonstrate ExM with apparent ~70-nanometer lateral resolution in both cultured cells and brain tissue, performing three-color superresolution imaging of ~10(7) cubic micrometers of the mouse hippocampus with a conventional confocal microscope.
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              SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.

              Meng-Tsen Ke, Satoshi Fujimoto, Takeshi Imai (2013)
              We report a water-based optical clearing agent, SeeDB, which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. Our method maintained a constant sample volume during the clearing procedure, an important factor for keeping cellular morphology intact, and facilitated the quantitative reconstruction of neuronal circuits. Combined with two-photon microscopy and an optimized objective lens, we were able to image the mouse brain from the dorsal to the ventral side. We used SeeDB to describe the near-complete wiring diagram of sister mitral cells associated with a common glomerulus in the mouse olfactory bulb. We found the diversity of dendrite wiring patterns among sister mitral cells, and our results provide an anatomical basis for non-redundant odor coding by these neurons. Our simple and efficient method is useful for imaging intact morphological architecture at large scales in both the adult and developing brains.
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                Author and article information

                Contributors
                Hana Kolesová
                Journal
                Journal ID (nlm-ta): iScience
                Journal ID (iso-abbrev): iScience
                Title: iScience
                Publisher: Elsevier
                ISSN (Electronic): 2589-0042
                Publication date PMC-release: 01 April 2021
                Publication date Collection: 23 April 2021
                Publication date (Electronic): 01 April 2021
                Volume: 24
                Issue: 4
                Electronic Location Identifier: 102387
                Affiliations
                [1 ]Institute of Anatomy, First Faculty of Medicine, Charles University, Prague, Czech Republic
                [2 ]Institute of Physiology, Czech Academy of Science, Prague, Czech Republic
                Author notes
                [∗ ]Corresponding author hana.kolesova@ 123456lf1.cuni.cz
                Article
                Publisher Item ID: S2589-0042(21)00355-2 Publisher ID: 102387
                DOI: 10.1016/j.isci.2021.102387
                PMC ID: 8086021
                PubMed ID: 33981974
                SO-VID: 82f08d1a-5ab9-4545-8451-e3348ae2c0e0
                Copyright © © 2021 The Authors
                License:

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                Categories
                Subject: Review

                Keywords: imaging methods in chemistry,optical imaging,biology experimental methods
                Data availability:
                Keywords: imaging methods in chemistry, optical imaging, biology experimental methods

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