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Diaphonization -- otherwise known as clearing and staining -- is one of the most photogenic preparation methods used by research scientists. It's beautiful, but is it practical? We interviewed Dr. Caleb McMahan, Collection Manager of Fishes, on how he uses this technique to answer questions about the evolution of fish!

To learn more about what's happening in Fishes at The Field Museum, check out their webpage: https://www.fieldmuseum.org/science/research/area/fishes

Big thanks to Caleb for taking the time to share his work with us, and to Alan Resetar for lending the reptile and amphibian specimens!... as well as the extra light table because ours broke right before filming. :(

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Producer, Writer, Creator, Host:
Emily Graslie

Producer, Editor, Camera, Graphics:
Brandon Brungard
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Filmed on Location and Supported by:
The Field Museum in Chicago, IL
(http://www.fieldmuseum.org)
[intro plays]

So we're here with Dr. Caleb McMahan, who's the collection manager of Fishes here at the field museum, and today we're going to be talking about clearing and staining. What is clearing and staining?

Caleb: So, clearing and staining is a process where we take an entire specimen and we clear that specimen down and use different stains to stain different parts of the skeleton.

Emily: So how is that process actually done -- how do you get from something that's, like, pickled in a jar like this, to something that you can literally see through?

Caleb: In general the process is that we start with a specimen, so here we have a species of cichlid from Nicaragua and Costa Rica and so we take this cichlid and the first thing we do -- and, again, some people do things differently, but I -- a lot of times will take the eyes out. If it's a really thick fish we might take the skin and scales off of it. And then we're going to take out the the guts. We take the gonads out and we store those in a vial back in the jar so that if later someone says I had the skeleton but I wanna see what sections of the reproductive tract look like they can still go back and do that.

So then we're left with our fish and we first dehydrate them, in really strong ethanol, like 95% ethanol, and then we start the process. And in general the two big phases. One, we're going to stain them so we're going to take the fish and we're going to use a stain called Alcian blue. And we're going to put the fish in this solution and it's gonna stain cartilage blue. And then we're going to then use the red dye called alizarin red. Things with calcium -- calcium phosphate -- will be stained red by this dye.

The clearing of it to clear away the tissue used trypsin which is an enzyme that digests proteins. It's found commonly in in vertebrate digestive tracts. And trypsin eats away at the at the tissue, the proteins, but it leaves the collagen. So you can see I can still pick this fish up. so it's still an actual specimen. --

Emily: It's like a jelly specimen!

Caleb: Right, right. It's still an actual fish specimen.. it's not just a pile of bones in the tray. And then we store it in glycerin, so this --

Emily: That's what the gooey, syrup stuff...

Caleb: The gooey kind of stuff. What's really neat about storing it in glycerin is that glycerin and collagen have the same refractive index. So, how light passes through - so when you put this on a tray on a light box like this you just get the skeleton.

Emily: So... that's how-- that's how we'll get invisibility cloak technology.

Caleb: Probably coming from clearing stained fish, yes.

Emily: Yeah, that will inspire how we develop that.

Why would you use this process over just something like -- you know, creating an x-ray or CT scan? It seems like it would be a little bit more involved than that.

Caleb: We still use x-rays a lot radiographs for looking at skeletons. But the nice thing about cleared in stained specimens is that we have a a 3d visualization of the skeleton, so X-rays don't give us that. Sometimes the location of certain cartilages or how they vary across different species or groups of species can tell you a lot about their evolution, their development, and anatomy. And in fishes a lot of times the larvae look nothing like the adults and so you can track how certain bones change through development, and how the general morphology of the fish changes -- and those make really interesting studies.

Emily: So you can try to essentially track the growth of an animal, or a species throughout different stages.

Caleb: Right, right.

Emily: Are fish the only thing that you clear and stain?

Caleb: They're not the only thing. So these are fish examples, but we also have a lot of examples from other vertebrate groups as well. These are amphibians and reptiles. So here we have a snake you can very nicely see the vertebral column wrapped up here. There's a frog, and the turtles are interesting because they look about the same size but you can see the blue one, there, is-- that's a cartilaginous skeleton, and so you've got an embryonic turtle there.

Emily: so that was a baby turtle.

Caleb: That's a baby baby turtle.

Emily: Aww.

Caleb: And then you have this smaller turtle -- the small turtle here with the red skeleton, that's, ah...

Emily: it's just a smaller -- small species of turtle, but it's an adult.

Caleb: Right.

Emily: so what are some of the discoveries or novel concepts that have been learned from using this process?

Caleb: in my own research I've used cleared and stained specimens to look at their teeth and morphology of their jaws, and how they look the same or different between species, or across groups of fishes.

Emily: So kind of how something could move in relation to another thing?

Caleb: Ah, or in their appearance, but that's that's another really, really interesting way that people are using cleared in stained specimens for biomechanics, and understanding -- I mean, even here we had this incredible diversity of morphology in fishes. And this particular species has this very long -- it's called an ascending process -- off of this upper jaw bone.

Emily: It's like a big extra bone up its forehead?

Caleb: Right, and so what this fish does, as you can see with the mouth closed --

Emily: Whoa!

Caleb: -- but to open the mouth you can use this specimen and you can see how the jaw opens.

Emily: Oh wow!

Caleb: Yeah, so using cleared and stained specimens allows us to look at how these things move and how these things feed.

Emily: so does this process destroy the specimen at all? Because it -- obviously it looks like it's kind of invasive.

Caleb: It doesn't destroy it -- it it changes the the the form of the specimen. Instead of looking like this, or like this, it's now a different appearance, and you can answer different questions with it. A lot of times I think, you know, we associate to tell different species apart or or groups of fishes -- that it's not all just always based on external anatomy or morphology. So, sometimes the characteristics of their of their skeleton which tell us what species it is. And- and so, sometimes - for instance - this is one of the type specimens of a knife fish. And so- so- we clear that this was cleared in stained as part of the description of the species to make sure that the skeleton -- and show how the skeleton -- was different from other species related to it.

Emily: Oh wow, so scientists don't don't see this process is destructive at all the informative.

Caleb: Yes, absolutely.

Emily: Can you clear and stain a human? Like, can you clear and stain me when I die?

Caleb: it would take a lot of trypsin.

Emily: It would be very expensive.

Caleb: And a lot of glycerin.

Emily: But you could do it.

Caleb: It would be very... expensive

Emily: Ok. I think that's a good use of scientific funds... probably not.

[end theme plays]

Emily: It still has brains on it.