TECHNICAL HISTORY
We owe the invention of transparentization to a German researcher, Werner Spalteholtz. In the 1900s, he and his team first came up with the idea of using chemicals to render tissue completely transparent. The technique has of course undergone a number of modifications since its inception, and has become very popular for imaging tissues, and even organs, in their entirety. Indeed, it is the pigmentation of the various cells that makes 3-D imaging difficult, since light cannot penetrate an opaque layer of pigments: and what light cannot penetrate is difficult to image with a microscope! Rendering a study object completely transparent by depigmenting it while retaining its shape, thanks to a strict protocol, is therefore extremely useful for reporting on the exact composition of a tissue or organ, which obviously provides crucial information on how it functions.
HISTOPARIS' CONTRIBUTION
The technique may be quite old, but making the eye transparent is something very new, and it's a technique perfected by the team! In fact, the main problem preventing the eye from becoming transparent is the presence of a layer of highly pigmented cells that protects the retina, the retinal pigment epithelium (known as the RPE). The RPE serves to completely isolate the eye from external light, so that the only ray of light captured is that which passes through the pupil. This ray of light is picked up by the fovea, the area at the back of the retina where the photoreceptor cells are located. If light weren't so precisely directed in the eye, we'd be perpetually dazzled and unable to see any sharp, high-contrast images !
However, the eye's ability to direct light was also the main difficulty in making it transparent. Nature's way: the RPE is so dense in pigments that depigmenting the layer without dissolving or destroying the other parts of the eye, which is a very fragile organ, is a real challenge !
HOW TO DO IT?
To make the eye transparent, several very precise steps must be followed. For the time being, only mouse eyes have been made transparent, but experiments are underway to make larger eyes transparent, with a view, of course, to making human eyes transparent. The protocol presented here has been simplified for ease of understanding: you can find the exact protocol in the Resources tab.
To transparentize mouse eyes, first fix them with chemicals to prevent the tissue from deteriorating, then store them in a formalin-based solution. The next step is to dehydrate the piece so that it can be bleached (i.e. stripped of its color pigments) without deteriorating it. However, simple dehydration would lead to deterioration of the object under study: that's why all the water has to be replaced by another chemical product. The part is therefore dehydrated in several successive baths, first to accustom the test object to the products, then to soak it so that all its water is removed.
After this, the part is bleached, then rehydrated in the same way, with several successive baths to rehab the part and rid it of all its chemicals. The part is then returned to a balanced environment, to prevent its natural deterioration. This is where we mark with fluorescent antibodies, using a technique called immunofluorescence, what interests us in the object we're studying.
As mouse eyes are too small to be imaged on their own, they have to be embedded in blocks of agarose, a gelatinous, transparent and solid product, which makes it easier to manipulate the objects without hindering their imaging.
Finally, we need to rid the object of the last thing that prevents us from imaging it perfectly: its lipids and fats, which don't retain light, but deflect it, which is also a handicap for transparencies. The eyes must therefore be incubated in tubes containing chemicals (make sure you use the right tubes, as some plastics are not resistant to the chemicals used! After all these steps, your eye should finally be properly transparentized ! All you need to do now is simply store it in a dark place.
