37: How tissue clearing - based 3D immunofluorescence allows for seeing more biology in the tissue w/ Sharla White, ClearLight Biotechnologies
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Even though tissues are tridimensional structures, most tissue research is done on two-dimensional tissue slides. This leaves a tremendous amount of biological information on the table. This episodes' guest - Sharla White, Ph.D., the vice president of research and development at ClearLight Biotechnologies )explains how tissue clearing and 3D immunofluorescence can take your tissue research to a whole new level. With the rise of immuno-oncology, the importance of immune cell interactions with the tumor cells is now routinely interrogated with immunofluorescent markers the spatial relationships of different immune cell populations are investigated. But how can we investigate something happening in a 3D space on a flat, two-dimensional tissue section? The truth is - in a very restricted manner. This is where tissue clearing and 3D immunofluorescence come into play. The tissue clearing technology -CLARITY, developed by ClearLight Biosciences allows for maintaining the integrity of tissue and visualizing cells in their original place and shape at the same time by using 3D immunofluorescence. In order to image deeper (beyond 100 micrometers), the light-scattering lipids of the tissue need to be removed and the refractive indexes of collagen, bone, and other tissue components need to be aligned. This is done after fixing the tissue and embedding it in a hydrogel. It ensures that the tissue structure is maintained before the detergent is applied to wash out the light-scattering lipids. Once tissue clearing is done, antibodies with properties and in amounts compatible with the process are used for 3D immunofluorescence.  This powerful technology does not come without challenges such as:

• the necessity of tissue bleaching for melanoma samples,
• selection of appropriate immunofluorescence markers,
• size of the 3D image files generated for visualization (often as big as 500 gigabytes reaching terabytes of data!)
• meaningful interpretation of the results

 Listen to the full episode to learn how Dr. White's team is approaching all the challenges, leveraging CLARITY potential and how this technology changes the way we do tissue research.  This episode's resources:

• ClearLight YouTube channel )
• ClearLight Biotechnologies website) 
• Original clarity paper "Structural and molecular interrogation of intact biological systems") 
• Imaris -  software for 3D visualization of tissue specimen )

Episodes you might also like:

• Immuno-oncology 101 w/ Elfriede Noessner, HelmholzZentrum, Visiopharm Advisor)
• Introduction to multiplex for tissue image analysis (Part 1) w/ Regan Baird, Visiopharm)

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