This blog post explores how high-resolution imaging enabled by tissue clearing technology will transform brain science and disease research.
The human brain is one of the most complex organs in the body. The number of neurons (the basic unit cells of the nervous system) in the brain is approximately 100 billion, comparable to the number of stars in our galaxy, the Milky Way, which contains our solar system. Thus, the human brain is composed of an immense number of cells interconnected in a complex manner, and its structure and function are not yet fully understood. Furthermore, the brain plays a central role in regulating higher-order cognitive functions such as sensation, memory, learning, and thought, and is crucial in forming human identity. Therefore, understanding how the brain works is akin to grasping the principles behind human thought processes, emotions, and behavior. To comprehend such a complex biological system, we must understand the characteristics and functions of the cells that compose the brain, as well as their arrangement and connection structures. In other words, a high-resolution 3D map—a blueprint of the human body at the cellular level—is required. We will explore tissue clearing technology, a groundbreaking technique gaining prominence in the creation of such high-resolution 3D maps.
Before examining what tissue clearing technology is, let’s look at the background behind its development. Numerous scientists have endeavored to create high-resolution human maps for tissue research. Traditionally, tissues like the brain have been imaged using methods such as Computed Tomography (CT), Magnetic Resonance Imaging (MRI), or Optical Coherent Tomography (OCT). These methods effectively enabled the three-dimensional representation of living human tissue structures, making them valuable for studying both structure and function. However, they suffer from limitations: their resolution is insufficient to reveal the characteristics of individual cells or their connection structures, and obtaining information at the molecular level is difficult. This is because the high density of cells creates a barrier that prevents light and chemicals from penetrating deep into the tissue. Particularly when cells are densely packed within human tissue, this lack of permeability makes it difficult to grasp intricate cell-cell interactions or neural network connectivity at a glance. Therefore, the current most advanced method for creating 3D maps is considered to be using high-resolution microscopes capable of observing down to the small cellular level within biological tissue.
However, when using high-resolution microscopy to observe large, opaque tissues like the brain, there is a limitation: the tissue must be cut into very thin sections, and each section must be imaged individually. This process requires reassembling thousands of 2D images into a 3D map, significantly reducing overall efficiency. Numerous studies have been conducted to address this issue, leading to the recent development of tissue clearing technology. In this context, the potential of tissue clearing technology is immense. It has opened the possibility of significantly refining basic research and diagnostics for various complex diseases like cancer, cardiovascular disease, and brain disorders through precise imaging at the cellular and molecular levels, in addition to life science research.
Tissue clearing technology is a method that renders opaque tissue transparent using specialized chemicals, paving the way to overcome the limitations of high-resolution microscopy. Among these, the recently developed CLARITY technique enables observation at a resolution approximately 2,000 times higher than magnetic resonance imaging. CLARITY technology synthesizes a transparent, porous polymer mesh called a ‘hydrogel’ within the tissue. This allows for the complete removal of lipids that make tissue opaque, while preserving tissue structure and molecules in three dimensions. Not only does it make the tissue highly transparent, but it also completely removes barriers that block the passage of light and molecular probes, allowing light and molecular probes to easily penetrate the tissue. Despite the complete removal of barriers like cell walls, the core of CLARITY technology is that the hydrogel preserves the tissue’s three-dimensional information at the molecular level, thus well-preserving the detailed morphology of cells and the interconnections between them.
Consequently, CLARITY-processed tissue becomes optically transparent, allowing light to penetrate deeply. This enables high-resolution microscopy imaging of thick tissues like the brain without the need for sectioning. Another advantage of CLARITY technology is the ability to image the three-dimensional distribution of specific molecules within the tissue. This is achieved by staining molecules covalently bound to the hydrogel with organic dyes. Furthermore, since the organic dyes can be removed without destroying the tissue structure or molecules, repeated analyses enable the study of various molecular phenotypes. This allows researchers to observe more vividly how cells and tissues interact in their living environment, and particularly to capture the patterns of change within tissues during the onset of specific diseases.
Furthermore, CLARITY technology can render most organs transparent, enabling 3D imaging. This makes it applicable not only to brain science but also to disease research in organs via histological examination. Notably, CLARITY also enables the real-time recording of the activity of specific disease-causing factors within organs and the resulting tissue responses. Therefore, the advancement of this technology is expected to significantly expand the scope of medical and scientific research and ultimately play a crucial role in the development of precision medical techniques.
However, the CLARITY technology is not without its challenges. Several issues remain to be resolved. The first challenge is accelerating processing speed. Unlike analyzing thin sections, processing large samples like mouse brains takes several months. This is because the process of deeply penetrating the necessary chemicals for tissue preservation, clearing, and staining is extremely slow. To address this, follow-up research is underway, and recently, domestic studies on ACT (Active Clarity Technique) have achieved results, demonstrating speeds up to 30 times faster than CLARITY. The second challenge is reducing costs. As the size of the tissue to be analyzed increases, not only are more compounds needed for clearing, but the amount of organic dyes, which are crucial for obtaining molecular information, also increases. Furthermore, the technical burden of processing large volumes of data remains a challenge to be addressed. Overcoming these limitations of low speed and high cost is essential for the commercialization of 3D mapping technology.
We have examined the background behind the development of tissue clearing techniques, the CLARITY technology, and the challenges it faces. What impact will this CLARITY technology have on humanity? Since Watson and Crick revealed the double helix structure of DNA, the Human Genome Project has now enabled us to decipher gene sequences and complete the human genetic map. Building on this, CLARITY technology will significantly contribute to completing a high-resolution map of the human body, providing answers about how cells expressing genes are interconnected. Scientists anticipate that the development of this technology will lead to the gradual unveiling of the brain’s secrets. Soon, a future will unfold where the structure and function of brain neurons are elucidated, enabling the treatment of intractable brain diseases like Alzheimer’s and Parkinson’s.
