enhanced 3d confocal imaging of solvent-cleared human heart tissues

Comprehensive 3D Analysis of Human Hearts from Micro to Macro Scales

As function follows form, understanding the architecture of the heart is fundamental for appreciation of its physiology. Although numerous investigations have revealed cardiac anatomy from micro- to macroscales1,2,3, multiple questions remain unresolved, especially those related to human cardiac anatomy. This is in part because basic studies focusing on functional anatomy generally utilized animal hearts4,5,6, which are often distinct from human hearts1,7,8. Furthermore, each individual study, even those using human heart samples, tends to focus on very specific structures, which renders it difficult to apply the findings in the context of the whole heart. This is even more so if the focused structures are at micro- or mesoscales, such as the perinexus9 and ganglionated plexuses10.
In this context, systemic structural study of the human heart rejected for transplantation provides a unique and rare opportunity to obtain a comprehensive atlas of cardiac structures in focus across microscopic and macroscopic scales11. Microscopic examination protocols include tissue clearing (modified immunolabeling-enabled three-dimensional (3D) imaging of solvent-cleared organs, iDISCO+)12,13, and immunohistochemical staining. Mesoscopic examination protocols include stereoscopic dissection, macro photography, and micro-computed tomographic (CT) scanning. Macroscopic examination protocols include gross dissection14, photography (including anaglyphs and photogrammetry)15,16,17, CT, virtual dissection18, and 3D printing of the physically or virtually dissected or whole heart17. In preparation for&#160;macroscopic examination, pressure-perfusion fixation is&#160;performed to maintain the 3D architecture and physiologically relevant morphology of the heart14,19,20,21.&#160;The combined application of these techniques is unique and crucial to correlate distinct anatomic features in the context of the 3D architecture of the human heart.
As the opportunity to obtain a non-pathological human heart sample is extremely limited, a multi-scale approach described herein maximizes the use of the sample. By applying various procedures described below, representative results will illustrate to the reader how the findings can be utilized for multiple purposes, including discovery in scientific research11 (comprehensive analyses of cardiac innervation, distribution of ganglionated plexuses), improvement of clinical procedures (simulation for surgical and interventional approaches), and anatomical education (real 3D demonstration of cardiac anatomy).

Author Spotlight: Advancing Human Cardiac Anatomy Through Multi-Scale Analysis of Hearts

Pipeline for Multi-Scale Three-Dimensional Anatomic Study of the Human Heart

Knowing cardiac anatomy is fundamental to understanding cardiac physiology. Studies of functional anatomy have spanned the micro to macro scales, but have largely used animal hearts. Systematic study of donor human hearts rejected for transplantation provides a unique opportunity to develop a comprehensive atlas that spans the microscopic to macroscopic scales.Through study of the human heart across different scales, we've demonstrated the exquisite innervation of the heart, identified cardiac anatomic structures to improve surgical and interventional approaches, and developed resources for anatomic education. The advantage of studying the human heart across different scales is that the detailed study of microscopic structures may be placed in the context of gross anatomy. The use of pressure profusion fixation maintains the physiologically relevant morphology of the heart.

Enhanced 3D Confocal Imaging of Solvent-Cleared Human Heart Tissues

To begin, use a scalpel to dissect 4%paraformaldehyde-fixed human heart tissue to fit within the chamber for confocal microscopy. Dehydrate the heart specimens in a graded methanol series for one hour each at room temperature with agitation, followed by overnight immersion in a 66%dichloromethane and 33%methanol solution with agitation. The next day, bleach the sample by performing the indicated steps.Then rehydrate the specimen with a graded methanol series for one hour each with agitation. After immunolabeling, perform the following steps to prepare the tissue for imaging. Affix a chamber containing adhesive to a slide and apply nail polish around the perimeter of the chamber.Turn on the upright laser scanning confocal microscope equipped with a 5X lens. Using laser lines appropriate for the emission spectra of the fluorophores used, acquire Tile scan and z-stack images at a resolution of 1024 by 1024. At the microscale level, tissue clearing of heart samples allows visualization of ganglia containing cardiac neurons and the neural patterning of myocardial innervation.

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Research

JoVE Journal

Biology

43 Views. To begin, use a scalpel to dissect 4%paraformaldehyde-fixed human heart tissue to fit within the chamber for confocal microscopy. Dehydrate the heart specimens in a graded methanol series for one hour each at room temperature with agitation, followed by overnight immersion in a 66%dichloromethane and 33%methanol solution with agitation. The next day, bleach the sample by performing the indicated steps.Then rehydrate the specimen with a graded methanol series for one hour each with agit...

Video: Enhanced 3D Confocal Imaging of Solvent-Cleared Human Heart Tissues

Detailed study of non-failing human hearts rejected for transplantation provides a unique opportunity to perform structural analyses across microscopic and macroscopic scales. These techniques include tissue clearing (modified immunolabeling-enabled three-dimensional (3D) imaging of solvent-cleared organs) and immunohistochemical staining. Mesoscopic examination procedures include stereoscopic dissection and micro-computed tomographic (CT) scanning. Macroscopic examination procedures include gross dissection, photography (including anaglyphs and photogrammetry), CT, and 3D printing of the physically or virtually dissected or whole heart. Before macroscopic examination, pressure-perfusion fixation may be performed to maintain the 3D architecture and physiologically relevant morphology of the heart. The application of these techniques in combination to study the human heart is unique and crucial in understanding the relationship between distinct anatomic features such as coronary vasculature and myocardial innervation in the context of the 3D architecture of the heart. This protocol describes the methodologies in detail and includes representative results&#160;to illustrate progress in the research of human cardiac anatomy.

This protocol presents a comprehensive pipeline to analyze samples obtained from human hearts that span the microscopic and macroscopic scales.

Detailed study of non-failing human hearts rejected for transplantation provides a unique opportunity to perform structural analyses across microscopic ...

Advanced Techniques in Cardiac Visualization from Gross Dissection to Virtual Reconstruction

After turning on a stereo microscope, perform delicate dissections on paraformaldehyde-fixed human heart tissue, focusing on structures such as the atrioventricular node, atrioventricular node artery, and cardiac nerve plexus. Place the heart sample on the scanner bed to acquire a CT image of the heart specimen. Using a 20 to 24 French surgical cannula, cannulate the superior vena cava for right heart perfusion.Then, occlude the inferior vena cava and another pulmonary artery with twine. Next, perform a progressive dissection of the heart tissue with photographic recordings at each stage of dissection. Using multiple light-emitting diode light panels set on C-stands with a wide black duvet and background cloth, photograph the heart with a digital single-lens reflex camera.Suspend the heart sample on a rotation table to obtain hundreds of multi-directional photographs with a smartphone. On Scaniverse and Polycam software, generate the 3D model of the heart in FBX format. Suspend the heart sample from a bar across the top of the container.Use a multi-detector row CT scanner to conduct CT scans of the heart. After generating virtual dissection images of the heart, visualize the non-enhanced walls, septa, and valves with virtual dissection to produce images similar to gross dissection.