How to Use Tissue Clearing and Volumetric Fluorescence Imaging

How to Use Tissue Clearing and Volumetric Fluorescence Imaging


In recent years, advancements in biomedical research have introduced innovative techniques such as tissue clearing and volumetric fluorescence imaging, revolutionizing our understanding of complex biological structures. These methods provide researchers with unprecedented insights into the three-dimensional organization of tissues, enabling a deeper exploration of cellular and molecular architectures. In this guide, we'll explore the basics of tissue clearing and volumetric fluorescence imaging, shedding light on their applications and benefits.

Tissue Clearing

Tissue clearing is a transformative technique that involves rendering biological tissues transparent, allowing for the penetration of light and improved imaging depth. This process addresses a major challenge in traditional microscopy, where the natural opacity of tissues limits the visualization of structures beyond the surface.

The procedure typically involves a series of steps to remove lipids and other light-scattering elements from the tissue. Researchers use specialized clearing solutions, which replace these components with substances that match the refractive index of the tissue. This matching refractive index reduces light scattering, resulting in improved transparency.

Commonly used clearing methods include CLARITY, Scale, and CUBIC, each with its own advantages and considerations. Researchers choose the method based on the specific requirements of their study, considering factors such as tissue type, size, and the desired level of transparency.

Volumetric Fluorescence Imaging

Once the tissue is successfully cleared, volumetric fluorescence imaging comes into play. This technique involves capturing high-resolution images of the entire cleared tissue volume, allowing researchers to explore intricate details in three dimensions. Unlike traditional two-dimensional imaging, volumetric fluorescence imaging provides a comprehensive view of the spatial organization of cells and structures within the tissue.

The concept of CLAM. Figure 1. The concept of CLAM. (Ren YX, et al.; 2020)

Fluorescent labeling is a crucial component of this imaging approach. Researchers introduce fluorescent dyes or proteins that selectively bind to specific cellular components, enabling the visualization of structures such as cells, organelles, and proteins under a fluorescence microscope. Multiple imaging modalities, such as confocal microscopy and light-sheet microscopy, are commonly employed to capture detailed images layer by layer.

Applications and Benefits

Neuroscience: Tissue clearing and volumetric fluorescence imaging have revolutionized our understanding of the brain's intricate neural networks. Researchers can now study the connectivity and organization of neurons in unprecedented detail, unraveling the complexities of neural circuits.

Developmental Biology: These techniques provide a unique perspective on embryonic development by allowing researchers to observe the dynamic processes of organ formation and tissue morphogenesis in three dimensions.

Pathology and Disease Research: Tissue clearing enables the comprehensive examination of disease-related changes within tissues. Researchers can explore the spatial distribution of pathological markers, aiding in the identification and characterization of diseases.

Drug Discovery: The ability to visualize drug distribution within tissues in three dimensions enhances drug development and testing. Researchers can observe how drugs interact with specific cellular targets and assess their effectiveness in a more realistic tissue context.

Challenges and Considerations

While tissue clearing and volumetric fluorescence imaging offer groundbreaking capabilities, researchers must be mindful of certain challenges. These include potential alterations to tissue structure during the clearing process, the choice of appropriate clearing methods for different tissues, and the need for specialized imaging equipment.


Tissue clearing and volumetric fluorescence imaging represent a paradigm shift in the field of biomedical research. These techniques empower researchers to explore the complexities of biological tissues in unprecedented detail, providing valuable insights across various disciplines. As technology continues to advance, the combination of tissue clearing and volumetric fluorescence imaging holds great promise for further discoveries, contributing to our collective understanding of the intricate world within living organisms.

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  1. Ren YX, et al.; Parallelized volumetric fluorescence microscopy with a reconfigurable coded incoherent light-sheet array. Light Sci Appl. 2020, 9:8.

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