2023-11-23 06:37:16
The three-dimensional structure of tissues and organs is of great importance in their functioning, and sometimes in certain pathologies. However, for practical reasons, biological analyzes are carried out on two-dimensional sections a few micrometers thick which are then passed under a microscope. The major obstacle to studying the organ in volume is the opacity of the tissues. However, for around ten years, there have been techniques to make these transparent. Fluorescent markers are then injected to obtain a 3D image of the organ. However, no protocol has yet made it possible to apply this approach to the entire eye. This is now done thanks to the work of Marie Darche, from the Quinze-Vingts national vision hospital, in Paris, and her colleagues.
Organs are opaque because they contain a wide variety of molecules with different refractive indices. A ray of light is therefore quickly diffused and does not penetrate the thickness of the fabric. To solve this problem, one idea is to standardize the refractive index. As early as 1914, German anatomist Werner Spalteholz used a method that began by dehydrating a mouse brain and then immersing it in a mixture of benzyl alcohol and methyl salicylate. The refractive index of this solution is similar to that of proteins, making the brain transparent to light.
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In 2012, Frank Bradke, then at the Max Planck Institute in Munich, was inspired by this almost century-old method. With his team, he developed a new one, called 3DISCO, which has disadvantages: it turns off the fluorescent markers and reduces the sample size by 30 to 40%. Other protocols were subsequently proposed, such as Clarity, uDISCO, etc. These processes standardize the refractive index and often get rid of membrane lipids responsible for light diffraction. These “transparization” techniques have had great success and make it possible to study, for example, the structure of an entire brain or of a mouse embryo.
The human eye was considered a difficult organ to explore using this approach. Indeed, its structure is very complex and heterogeneous, with numerous tissues, some of which are highly pigmented or fragile, such as the retina. Until now, imaging had been carried out by isolating certain tissues (the sclera, for example), but not on the entire eye.
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Marie Darche and her colleagues have developed an eye transparency protocol, called ClearEye, which is an adaptation of previous protocols to the specificities of the organ of vision. The entire preparation process takes more than thirty days. In addition to making the organ transparent, the researchers found a way for the fluorescent antibodies to penetrate deep into the tissue (they were limited to 1 or 2 millimeters until now). To obtain precise images, the team then used a microscope specializing in large samples, known as a “light sheet” microscope, from the Wyss research center in Geneva, Switzerland.
Anterior segment of the eye observed using a transparization method with double marking. Here the purple traces actin, which plays a role in the contraction of muscles and arteries, which makes it possible to distinguish the latter from veins.
© Marie Darche
In this study, researchers benefited through organ donation from four healthy human eyes. They were thus able to validate the method and establish the overall architecture of the blood vessels and ocular nerves.
“Since the release of our study, we have digitized more than fifteen whole human eyes,” explains Marie Darche, “and we have started work on the first pathological eyes. By digitizing a large number of healthy and pathological organs, we want to create three-dimensional eye banks. These high-precision images will be shared with both researchers and doctors. » These images will notably provide a rich and powerful tool for studying pathologies that are often multi-tissue or which sometimes affect the eye as a whole, such as AMD (macular degeneration due to age), glaucoma, retinal vein occlusion (RVO), uveal melanoma… and the causes of which are not always well understood.
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