Name: mesoscopic optical imaging of whole mouse heart
Uploaded: 2021-10-14T14:15:00
Description: Watch this Scientific Journal Video about Mesoscopic Optical Imaging of Whole Mouse Heart at JoVE.com

This project has received fundings from the European Union&#8217;s Horizon 2020 research and innovation programme under grant agreement No 952166 (REPAIR), MUR under the FISR program, project FISR2019_00320 and Regione Toscana, Bando Ricerca Salute 2018, PERCARE project.

All animal handling and procedures were performed in accordance with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and conformed to the principles and regulations of the Italian Ministry of Health. The experimental protocol was approved by the Italian Ministry of Health (protocol number 647/2015-PR). All the animals were provided by ENVIGO, Italy. For these experiments, 5 male C57BL/6J mice of 6 months of age were used.
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In this work, a successful approach to clear, stain, and image a whole mouse heart at high resolution was introduced. First, a tissue transformation protocol (CLARITY) was optimized and performed, slightly modified for its application on the cardiac tissue. Indeed, to obtain an efficient reconstruction in 3D of a whole heart, it is essential to prevent the phenomenon of light scattering. The CLARITY methodology allows us to obtain a highly transparent intact heart, but it requires long incubation times when performed pas...

Structural remodeling associated with cardiac diseases can affect electrical conduction and introduce electromechanical dysfunctions of the organ1,2. Current approaches used to predict functional alterations commonly employ MRI and DT-MRI to obtain an overall reconstruction of fibrosis deposition, vascular tree, and fiber distribution of the heart, and they are used to model preferential action potential propagation (APP) paths across the organ3,4. These strategies can provide a beautiful overview of the heart organization. However, their spatial resolution is insufficient to investigate the impact of structural remodeling on cardiac function at the cellular level.
Placing this framework at a different order of magnitude, where single cells can play individual roles on action potential propagation, is challenging. The main limitation is the inefficiency of standard imaging methods to perform high-resolution imaging (micrometric resolution) in massive (centimeter-sized) tissues. In fact, imaging biological tissues in 3D at high resolution is very complicated due to tissue opaqueness. The most common approach to perform 3D reconstructions in entire organs is to prepare thin sections. However, precise sectioning, assembling, and imaging require significant effort and time. An alternative approach that does not demand cutting the sample is to generate a transparent tissue. During the last years, several methodologies for clarifying tissues have been proposed5,6,7,8&#8288;. The challenge to produce massive, transparent, and fluorescently-labeled tissues has been recently achieved by developing true tissue transformation approaches (CLARITY9&#8288;, SHIELD10&#8288;). In particular, the CLARITY method is based on the transformation of an intact tissue into a nanoporous, hydrogel-hybridized, lipid-free form that enables to confer high transparency by the selective removal of membrane lipid bilayers. Notably, this method has been found successful also in cardiac preparation11,12,13,14&#8288;. However, since the heart is too fragile to be suitable for an active clearing, it must be cleared using the passive approach, which requires a long time to confer complete transparency.
In combination with advanced imaging techniques like light-sheet microscopy, CLARITY has the potential to image 3D massive heart tissues at micrometric resolution. In light-sheet microscopy, the illumination of the sample is performed with a thin sheet of light confined in the focal plane of the detection objective. The fluorescence emission is collected along an axis perpendicular to the illumination plane15. The detection architecture is similar to widefield microscopy, making the acquisition much faster than laser scanning microscopes. Moving the sample through the light sheet permits obtaining a complete tomography of big specimens, up to centimeter-sized samples. However, due to the intrinsic properties of the Gaussian beam, it is possible to obtain a very thin (of the order of a few microns) light-sheet only for a limited spatial extension, thus drastically limiting the field of view (FoV). Recently, a novel excitation scheme has been introduced to overcome this limitation and applied for brain imaging, allowing 3d reconstructions with isotropic resolution16.
In this paper, a passive clearing approach is presented, enabling a significant reduction of the clearing timing needed by the CLARITY protocol. The methodological framework described here allows reconstructing a whole mouse heart with micrometric resolution in a single tomographic scan with an acquisition time in the order of minutes.

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mesoscopic optical imaging of whole mouse heart

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

The developed passive clearing setup allows to obtain a cleared adult mouse heart (with a dimension of the order 10 mm x 6 mm x 6 mm) in about 3 months. All the components of the setup are mounted as shown in Figure 1. The negligible temperature gradient between each clearing chamber (of the order of 3&#176;C) allows maintaining the temperature in a proper range across all chambers.
<img alt="Figure 1" class="xfigimg" src="/files/f...

Watch this Scientific Journal Video about Mesoscopic Optical Imaging of Whole Mouse Heart at JoVE.com

Mesoscopic Optical Imaging of Whole Mouse Heart

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition ...

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