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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(139), e57769 (2018).</li><li>Olianti, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+imaging+and+morphometry+of+the+heart+capillary+system+in+spontaneously+hypertensive+rats+and+normotensive+controls.">3D imaging and morphometry of the heart capillary system in spontaneously hypertensive rats and normotensive controls.</a> Scientific Reports. 10, 1-9 (2020).</li><li>Pianca, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+sympathetic+innervation+network+shapes+the+myocardium+by+locally+controlling+cardiomyocyte+size+through+the+cellular+proteolytic+machinery.">Cardiac sympathetic innervation network shapes the myocardium by locally controlling cardiomyocyte size through the cellular proteolytic machinery.</a> The Journal of Physiology. 597, 3639-3656 (2019).</li><li>Di Bona, A., Vita, V., Costantini, I., Zaglia, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+clearer+view+of+sympathetic+innervation+of+cardiac+and+skeletal+muscles.">Towards a clearer view of sympathetic innervation of cardiac and skeletal muscles.</a> Progress in Biophysics and Molecular Biology. 154, 80-93 (2020).</li><li>Voigt, F. 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Nothing to disclosure.

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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			<td>2-2&#8217; Thiodiethanol</td>
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			<td>Nikon</td>
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			<td>Extera&#236;nal quartz cuvette</td>
			<td>Portmann Instruments</td>
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			<td>Thorlabs</td>
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			<td>M-122.2DD</td>
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			<td>Sigma-Aldrich</td>
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			<td>Thorlabs</td>
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			<td>Agar Scientific</td>
			<td>R1018</td>
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			<td>Phosphate Buffer Solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
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			<td>Polycap AS</td>
			<td>Whatman</td>
			<td>2606T</td>
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			<td>Relay lens</td>
			<td>Qioptiq</td>
			<td>G063200000</td>
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			<td>Sigma-Aldrich</td>
			<td>L3771</td>
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			<td>Thorlabs</td>
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			<td>KNF Neuberger Inc</td>
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			<td>Water bath</td>
			<td>Memmert</td>
			<td>WTB</td>
			<td></td>
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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 ...

This methodology combines improvement of clearing protocol with the eye capability of optical sectioning of the Mesos PIM technology allowing us to perform a meso scopic reconstructions at micrometric resolution. It provides the possibility to image massive cardiac tissues or entire mouse hearts entire solution in a single scan without losing the original three-dimensional tissue organization. After the heart has been cannulated by proximal aorta washed in tyro solution and fixed in 4%Paraform aldehyde in 0.01 molar PBS it is ready to start the clearing protocol.Wash the heart three times in 0.01 molar PBS at four degrees Celsius for 15 minutes. After this step, the heart can be stored in PBS and 0.01%sodium azide at four degrees Celsius for several months. Incubate the heart in 20 milliliters of hydrogel solution in shaking condition at 15 RPM for three days at four degrees Celsius.De-gas the sample at room temperature using a dryer a vacuum pump, and a tube system that connects the dryer to both the pump band and nitrogen pipeline. Place the sample in the dryer and open the vial keeping the cap on it. Close the dryer and remove the oxygen from the tube by opening the nitrogen pipeline.Turn on the vacuum pump to remove the oxygen from the dryer for 10 minutes. Turn off the pump and open the knob of the dryer to the nitrogen pipeline. Then open the nitrogen tap.Once the pressure is equal to the atmospheric pressure carefully open the dryer and quickly close the vial. Keep the heart in the De-gased hydrogel solution at 37 degrees Celsius for about three hours at rest. When the hydrogel is properly polymerized and appears entirely gelatinous, carefully remove the heart from it and place it in a clearing solution in the vial with clearing solution.Change the clearing solution in the vial once every three days to speed up the clarification procedure. Once the heart appears completely clarified remove it from the vial and wash it in 50 milliliters of warmed up PBS for 24 hours in shaking condition. Wash again in 50 milliliters of one X PBS T for 24 hours in shaking condition.Incubate the sample in 0.01 milligrams per milliliters wheat germ aglutinin Alexa floor 633 in three milliliters of one X PBS T in shaking condition at 50 RPM at room temperature for seven days. After the seven day incubation, wash the sample in 50 milliliters of one X PBS T at room temperature in shaking for 24 hours. Incubate the sample in 4%PFA for 15 minutes and then wash it three times in 0.01 molar PBS for five minutes each.Incubate the heart successively in 20 and 47%TDE in 0.01 molar PBS for eight hours each. And then finally at 68%TDE in 0.01 molar PBS to provide the required refractive index of 1.46. Gently fill about 80%of the external quartz quvet with the refractive index medium.Fill the internal quartz quvet with the same refractive index medium. Immerse the sample inside the internal quvet. Gently move the sample to the bottom of the quvet using thin tweezers and arrange the heart with its longitudinal axis parallel to the quvet's main axis to minimize the excitation light path across the tissue during the scanning.Carefully fix the tailored plug above the internal quvet with two screws. Mount the sample to the microscope stage using magnets. Translate the vertical sample stage manually to immerse the internal quvet into the external one.Turn on the excitation light source with a wavelength of 638 nanometers and set it at low power in the order of three milliwatts. Move the sample using the motorized translator to illuminate an inner plate of the tissue. Turn on the HC image live camera software and set the camera trigger on external edge trigger light sheet mode to drive the acquisition trigger of the camera by the custom software controlling the entire setup.Enable auto save in the camera software and set the output folder where the images need to be saved. Manually adjust the sample position in the X Y plane with the linear translators to move the sample to the center of the field of view of the camera sensor. Move the sample along the Z axis using the linear motorized translator to identify heart borders for tomographic reconstruction.Increase the laser power to approximately 20 milliwatts before beginning for the imaging session. Start the tomographic acquisition by clicking the start button in the capture panel of the imaging software, and at the same time start moving the sample along the Z-axis at the constant velocity of six micrometers per second using the motorized translator. The combination of the clarity methodology with TDE as refractive index medium does not significantly change the sample's final volume nor leads to an isotropic deformation of the specimen.The excitation optics produced a light sheet with a minimum waste of about six micrometers that diverged up to 175 micrometers at the edge of the field of view. The synchronization of the camera rolling shutter with the axial scan of the light sheet waste ensured to collect the emission signal only in the sample portion excited with the waste of the light sheet resulting in an average F W H M of about 6.7 micrometers along the entire field of view. The Z point spread function of the microscope was also estimated by a tomographic reconstruction of the fluorescent nanosphere and an F W H M of 6.4 micrometers was estimated by the fit.The potentiality of the system to resolve single cellular membranes in three dimensions with a sufficient signal to noise ratio in the entire organ was also confirmed. The de-gassing procedure is the most critical step of the protocol. If it is not properly performed the tissue could encounter damages indicating during the incubation in a cleaning solution.The presented protocol can be combined with a multi staining protocol to achieve a whole organ reconstruction integrating different biological structures and can be applied to study pathological models.

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1.8K visualizações.  European Laboratory for Non-Linear Spectroscopy. Esta metodologia combina a melhoria do protocolo de limpeza com a capacidade ocular de seccionamento óptico da tecnologia Mesos PIM, permitindo-nos realizar reconstruções meso scópicas em resolução micrométrica. Ele fornece a possibilidade de visualizar tecidos cardíacos maciços ou toda a solução de corações de camundongos inteiros em uma única varredura sem perder a organização tridimensional original do tecido. Após o coração ter sido canulado por aorta proximal lavada em...