The clearish lump looks like some bizarre, translucent gummy candy that might have once been piña colada flavored. But this is something you definitely don’t want to eat. That see-through blob was once a mouse.
In the Aug. 14 Cell, scientists at Caltech detail the difficult series of methods required to make small animals such as mice and rats completely translucent. Scientists have been trying to clear tissue for better observation since the 1800s, and, as with all science, the new techniques build on many previous experiments done in a variety of labs. The techniques make it possible to capture images both beautiful and gross. And the procedure will teach scientists more about anatomy than ever before.
If you want to render an animal transparent, you first have to overcome a solid problem: lipids. This group includes molecules essential to life, such as fats, cholesterols, waxes and steroids. Lipids form the membranes that surround our cells, the hormones that make us grow and reproduce and much, much more. But lipids have a problem. You can’t see through them.
So to render an organism transparent, you need to remove the lipids. Bin Yang and colleagues in Viviana Gradinaru’s lab at Caltech used detergents to dissolve the lipids. The technique is based on CLARITY, a method that Gradinaru helped to develop in Karl Deisseroth’s lab at Stanford. Scientists there rendered a mouse brain transparent using CLARITY.
Gradinaru explains that for a clear organ, dissolving lipids alone alone isn’t enough. “Without lipids the tissue would just collapse, so we need to maintain the structure of the tissue,” she says. They did this by infusing acrylamide, a chemical that links proteins and other molecules in the tissue together, forming a gel. This means that they can dissolve the lipids and leave the structure intact. Gradinaru’s group called this technique PACT, for Passive CLARITY. It does take a while. Dissolving out the lipids in a whole mouse or rat takes one to two weeks.
The choice of detergent is crucial in this process. Many other clearing techniques produce beautifully transparent organs. But the detergents often dissolve other chemicals as well, including fluorescent markers. Fluorescent tags can be used to label cells or specific proteins during an experiment to compare different conditions and observe change. Scientists need those labels to remain nice and bright in a cleared specimen. Previous techniques struggled with trying to find a clearing detergent that would efficiently render all the organs of the body transparent, while leaving the fluorescent tags alone. CLARITY was one of the best, leaving tags intact while clearing out a whole brain.
There’s also the sheer amount of time these processes take. A single organ is small, and detergent can be diffused through relatively quickly. But a whole body, even a small mouse body, could take far too long. Gradinaru and her group realized that the best option is perfusion. This involves pushing a chemical (such as your detergent) through the circulatory system of the animal under continuous pressure. The circulatory system runs all through the body, so the detergent gets everywhere. The scientists call this method PARS, for perfusion-assisted agent release.
Once the tissue has been made transparent, Gradinaru’s lab embeds it in a solution that makes it easily viewed and photographed. The end result is a see-through mouse or rat that can be labeled with fluorescent dyes. The tissue is so clear that the researchers have been angle to detect single strands of RNA, Gradinaru says. They have also used the technique to isolate and label cancer cells from a human skin cancer biopsy.
Gradinaru has high hopes for the method. “We can obtain better maps for neuronal networks,” she says. “A transparent whole organism is particularly useful to map the distribution and identity of peripheral nerves at their target organs.”
Mapping the final endpoints of nerves in detail could produce better methods for electrical stimulation, which could aid in therapies for disorders such as migraine, epilepsy and depression. Deisseroth also hopes that the technique can aid in “studying whole-body progression of cancers” and give scientists a better understanding of how mammals develop and age.
While clear mice and rats can provide amazing detail, they also have limits. The main limitation: The technique requires a dead mammal. “Because all the tissue is postmortem we cannot investigate phenomena as they happen in real time — we can just get snapshots,” Gradinaru explains. Being able to clear out a living animal would allow scientists to see whole bodies, functioning all the way down to the molecular level, in real time.
A fully transparent live mouse or rat (and its accompanying horror movies) is probably a long way in the future. Even so, the current technique allows scientists a better view of the body than ever before. That bizarre gummy-looking mouse may look strange, but it represents more new and impressive opportunities to enhance our understanding of our bodies and ourselves.