Best of: The future of transparent tissue

0:00Hey everyone, it's your host Russ Altman from the future of everything here. About a year ago, a research team at Stanford Engineering led by Guo Sung-hong published a paper about their work to use a common food dye to make mouse skin transparent. They're now trying to apply this to humans, and their findings made a big splash and have a potential to provide a range of benefits in health care. You can imagine that if we have the ability to see what's going on under the skin without having to cut it or use radiation to get a clear look, this could improve lots of things, including biopsies and blood draws.

0:32I hope you'll tune in again and enjoy. Before we get started, another reminder to rate and review, particularly if you learn something new or find this podcast to be stimulating.

0:50Well, you know, you look at a living organism and it is not see-through. It is not transparent. Sometimes when we were kids, there were those models made out of plastic where you could see through the transparent skin and then see the guts, the heart, the lungs, the intestines. Well, guess what? Scientists have figured out how to make tissue, living tissue and also dead tissue, how to make that see-through. It's related to a complex physical phenomenon where by manipulating the physical properties of both lipid and the water that it interacts with. Lipids are fats, water is water, they interact and that often causes the cloudiness that leads to not being see-through.

1:32Well, Guo-Sung Hong from Stanford University is an expert in physics, material science, and biology, and he and his colleagues have figured out how to add something to the water so that it's similar to the lipid and it all looks see-through. It first worked on a chicken breast and then they moved to mice and it's an amazing story. Guo-Sung, you're a specialist in material science, and yet you work on biology and the brain, which might not be the first thing that people think of when they think about materials. So can you explain how do you bring these things together and what's your interest? So my interest actually started from when I was a graduate student at Stanford, I actually was working on using some very exotic nanomaterials.

2:16These are called carbon nanotubes that have very interesting emission properties that allow us to see deep into the brain. So that's all my interest in brain science started. And I moved to Harvard to continue my post-opo research. And then during that time, actually, I had the privilege of working on using flexible materials for brain-machine interfaces that actually further strengthened my knowledge and interest in using material science for brain science. And then coming back to Stanford, now my lab is mainly focused on developing new tools for minimally invasive imaging and neuromodulation, well, apparently also for neuroscience and the body in general.

3:01Great. Now, one of the things that has gotten a lot of people very excited is this work. I don't even know how to characterize it, but making some tissues be like see-through, like transparent. And so this is amazing. And I know it's very complicated, but I wanted to try to understand how it works. So can you take us through what is the technical reason for even doing this for the science? And then what do we need to know about the physics to understand how this works? Yeah, sure. So the motivation behind this work is actually for me, for us, is actually the challenge. This is actually a quite universal challenge that is present for all light-based methods using biomedicine.

3:43So we know that the light actually does not penetrate very deep into tissue. So this can be tested by just looking at your hands. You can see the surface features, but you cannot really see it through. That's because our body actually made of, you know, basically it's a giant bag of water, 70 % water, but it also has a lot of teeny tiny lipids structures such as the membranes and also all the organelles that are made of lipids and also proteins. And all these components are actually having a refractive index much higher than water. So now when we have components of the tissue with different refractive indices mixed at the microscopic scale, it necessarily makes the tissue actually scatter the light as the light goes through.

4:24So then the scattering really limits a lot of the applications, you know, in biomedicine. For example, we use fluorescent imaging to understand, to visualize the structures and understand, to study the activity and the function of biology. But fluorescent imaging typically works the best for an ex-inviable piece of tissue, which you can get perfect resolution. But when we're talking about inviable animals, it's actually quite challenging because how are you going to get a light in and out to, let's say, millimeters of tissue, let alone the entire body of the human? Right. So what you're saying is that it's great if you just want to start at the surface, but anything deeper, you would either have to cut them open, which is what we do, what surgeons do, biologists, or come up with a very amazing technology.

5:09Exactly. So what we wanted to hope to achieve is that we don't have to cut open tissue. We don't have to insert enough fiber. We don't have to insert a microendoscope, but we'll be able to make the tissue transparent by itself. And actually, apparently, we're not the first to tackle this challenge. There has been amazing work done in the past decade where people looked at various transparency technologies. These are called tissue clearing. So, you know, a lot of really amazing work done in this field. So, conventionally, for tissue clearing, we have to either remove lipids, these are the high index components from this water bag of the body, or we have to replace water with high index components, such as mixing the tissue with organic solvents such that the tissue in the water is replaced to match the index up to the lipid and protein.

6:00Apparently, as you have seen, either approach will necessarily have to remove some vital components that are vital to maintaining life. Right, right. So just to repeat back, the water, if you remove the water and replace it with an organic solvent, that tissue is not going to be functioning normally in the future. and then so you okay gotcha keep going yeah so then what we wanted to understand is that can we find a way to not having to replace either water or lipids or something else but maintaining well but actually achieving the two transparent okay so that requires us to look at why tissue why water and lipids have different refractive indices it turns out that water and lipids despite being transparent in the colorless in the visible spectrum they are actually very strongly absorbing light in the deep UV and this actually absorption ultraviolet Ultraviolet exactly okay, and this give you the absorption after about it absorption is the reason why they have different refractive indices Invisible this is a kind of like a mind-boggling concept because we typically think that Different wavelengths are different colors are basically independent from each other when we're talking about red light We don't mix it up with green or blue Right in fact all wavelengths are actually a causally connected via some kind of deep you know, underlying physical and mathematical principle, which is known as the Quimus-clonic relations, which basically tells us the absorption at one wavelength is going to determine the refractive index of different wavelengths and vice versa.

7:31So that basically tells us water and lipids have different refractive indices because they derive their different refractive indices from the different absorption in the outer body. And along the similar line, we could come up with the engineering principle by making water more absorptive in the uv or in a shorter region of the visible spectrum such that water will be as refractive as lipids at a longer wavelength such as the red spectrum of the visible right so let me make so this is now getting into physics and i just to keep it uh accessible what you're saying is that what we would like is to match the performance the refractive index the the color so to speak of water and lipids in the visual range But in order to do that, we have to manipulate how they respond to UV light.

8:16Exactly. That's right. Which is amazing. And we're just going to say it's a miracle of physics that this is a relationship that might not have been taken advantage of previously. Exactly. That's right. Okay. So now then, how do we do this? So, yeah, that actually, you know, this really opens up a lot of opportunities for doing this. So one of them is if we can identify really absorbing, strongly absorbing such as dye molecules with absorption in the UV, absorption in the shorter range of the visible spectrum, such as in the blue or violet region, then it will have the effect of raising the refractive index of water at a longer range of the visible spectrum to match the refractive index of lipids without changing the chemical properties of water.

8:56And this is a dye that you would dissolve into the water, is that right? so that it changes the overall properties of the solution that you're looking at. Exactly. That's right. Wow. Okay. So does it work? Yes, it do work. Actually, you know, I have a team of very talented post-option graduates. They actually did a screening of many, many dyes. And it turns out one of the dyes, which is actually used in the food industry, this is a tatrazine. A lot of people also call this yellow number five. This is actually used in, you know, snacks such as the Doritos in here. Oh, one of my favorites. So to the naked eye, it looks orange?

9:33To the naked eye, it looks orange slash red. Okay. Yeah. And then, wow. Okay, keep going. This is amazing. So Doritos is the answer to transparency. Yeah, exactly. So Doritos' eye, the transparency looks red because it absorbs light in the blue region, which is actually very short wavelength, around 430 nanometers. And then this absorption, these two, the refractive index change upon dissolution in water at a longer wavelength, which is the red, which is actually beyond 600 nanometers. And then basically, what we have demonstrated is that the aqueous solution of the little zye has the refractive index similar to that of lipids.

10:12Okay, and that was the goal, was to try to get them matched. Exactly. That's right. Okay, so what were the first experiments? Like when you said screening, you're obviously not doing this in humans. What kind of tissue do you use and what kind of transparency did you get? Yeah, that's actually a good question. So actually, first of all, we used a set of optical characterization methods, such as the UV visible absorption spectrometer, as well as the spectroscopic elapsometer. And these are basically very fundamental optical characterization tools. And then once, you know, upon identifying some top containers, top candidates of these dye molecules, we then apply this solution of the dyes to a piece of chicken breast.

10:53The chicken breast, there you go. Yeah, the chicken breast, you know, it actually looks pinkish and it doesn't really, it's opaque, it doesn't allow the light to pass through. But then we found that if we take the chicken breast and soak that into the deridole's eye solution, just within nearly a few minutes, we can see the transparency significant changes. It becomes, it goes from complete opacity to translucency. And if you let it in for even longer, let's say half an hour to an hour, it becomes completely transparent. and then it achieves the transparency close to 80 90 okay now okay we have to stop because this is amazing first of all how thick is the piece of chicken breast like i'm sure it's not a whole chicken breast or is it right yeah so we what we have done is about one to two millimeters in thickness which is not very thick uh but actually the nsf national science foundation they actually developed a uh a protocol so for any interested you know you know uh students you know as a kids to the producers at home.

11:49And then they actually get something that is as thick as five millimeters. Okay, and now you're getting to be like, that's a thick piece of paper or a very thin piece of chicken. So you slice that and you simply soaked it and you counted on the fact that the water with the dye would diffuse by natural processes into the chicken and replace the, I'm calling it the normal water. And as that happened, it became see-through. Right, exactly. It becomes... Unbelievable. Okay. So, okay, now I'm imagining you're pretty excited. How have you taken this further? Like, what are the serious applications of this?

12:28Yeah, so actually it really opens the door to many, many different applications. One of the things we're doing, we're very, very excited about is actually we're working with dermatologists at this time. And we're actually working on the human skins. These are actually freshly dissected human skins with the goal of one day applying this to the human patients. And then the data is actually very, very promising. We're about to publish a paper in an upcoming paper. And then what we have found is that there are many different dye molecules, well, apparently the derido dyes included, that has the effect of making the human skin transparent as well.

13:06And then the human skin, in principle, is actually much thicker than the MOST skin that we have tested in the paper. So having a human skin transplant really has a lot of applications. For example, we'll be able to, if this can be used for human patients, then we might be able to identify some kind of medical conditions, such as, let's say, basal cell carcinoma. Yes, skin cancer, very common, and maybe would look different when you could see through. Yeah, exactly. That's right. So then we can actually detect these structures in the deep tissue. And this is one of the applications. And then we're also combining this technology with a variety of imaging modalities used commonly in lab animals.

13:50For example, we're doing two full-time, three full-time microscopy. We're actually applying this combined with a light sheet microscopy. And interestingly, actually, we're very delighted to see quite a few labs very quickly picked up, you know, they adopted this technology and applied that in their own systems. For example, a group, Adam Watt's lab at Duke University, they actually applied this to OCT, the optical coherence tomography, and they realized that they could actually enhance the penetration depth by two to three times in the live animal. Okay, so what I'm hearing is that many of these other technologies that would have been only applicable on the surface until the light gets scattered, by removing that, effectively removing that layer they can get better data on the deeper layers of tissue exactly so i before we go on i just i was thinking about the human and the dermatology and we went from chicken breast to humans now the chicken breast is dead and we don't really care what we do to the chicken breast make it we might make it a little bit saltier and tasty but separate from that because of the doritos um what is the impact when you're doing this can you do this on a live tissue and does it still work and are you doing damage to the tissue i'm sure you're worried about this and doing a lot of tests in that area.

15:02Exactly. Yeah. So actually, we also, that's an excellent question. We did this on live animals, so live mice, basically. So what we have shown is that we could apply this to the shaved mouse abdomen. And then typically, the shaved mouse abdomen, actually, it has a skin, it has a connective tissue, it has fat, it has muscles. So all of this actually would make the abdominal wall not transparent. We cannot see through the abdomen to visualize the inner organs. but what we have found that is quite striking to us when we first saw this is that by applying dye molecules dissolving water onto the surface of the skin abdominal skin we could actually visualize liver intestines uh you know even bladder and a lot of different organs the inside the mouse uh while the mouse is still alive we could actually see the mouse heart you know beating you can see the lungs breathing we can even see the gut moving that's unbelievable we're looking at the motility of the gut through the uh transparentized uh mouse abdominal wall when um when you do it to a living mouse uh of course we know that the skin gets circulation so it's going to be clearing it's going to be clearing the water or the the dyed fluid is going to i'm guessing is slowly going to dissipate as it gets brought to other parts of the body so is that an issue and do you have to kind of refresh it or in some way that's right that's actually a very good point Actually, if you just leave the dye in there, it probably will maintain that effect for about maximum of 30 minutes.

16:29But after that, the effect gradually gets washed out because of the clearing of the body, as you mentioned. So that requires us to apply this again. So what is nice about this technology is actually transient and reversible. Getting back to your point about the safety of this. Apparently, we actually support the Doritos dye. It is not the best dye that potentially we can find. We're still actually looking for other more efficient, more potent dyes. But the little dye actually will still have to use a pretty high concentration after this. And that concentration, if you leave it on a stain for too long, it may cause some kind of adverse effect.

17:03And then currently what we're doing is actually we leave this transparency window for about maximum 30 minutes. And after that, we'll have to wash out dye by using salient to extract the dye molecule. And then we saw that. We actually observed that the stain actually very nicely returned to the original opaque state. now i'm going to ask one ridiculous question i always allow myself one ridiculous question when i eat doritos and it's all over my tongue am i making my tongue slightly translucent that's actually a very good point actually yeah you know based on the physics yes it will make some of the probably epithelial cells slightly transparent but the thing is that people probably have never used a microscope to look at how to characterize that kind of transparency but i believe a confidates that is indeed happening.

17:51You're listening to The Future of Everything, and we'll have more with Guo Song Hong next.

18:07Welcome back to The Future of Everything. I'm Russ Altman, and I'm speaking with Guo Song Hong from Stanford University. In the last segment, we learned how Guo Song and his team figured out how to make tissue transparent. We're going to discuss now whether this occurs in nature, and then we'll move to another project where he's figured out how to introduce light into the body without any wires or electricity. In this section, I wanted to ask more generally, is this transparency phenomenon something that occurs in nature? Yeah, it's actually an excellent question. So it turns out that many species, as you already know, these are aquatic species, not in mammals.

18:45So these species are actually derived there. They also are inherently transparent. These species such as zebrafish larvae and also glass frogs, they derive these huge transparency. So zebrafish and a certain type of frog. They're larvae, but I've seen full-grown zebrafish and they retain a certain amount of transparency. Oh, that's interesting. Okay, so I need to update my time. A little bit, yeah. So I read some papers recently and it turns out that people only in the recent years they started realizing that some of these inherently transparent species derive their the high transparency in a tissue from the high refractive index components.

19:28These are usually proteins in the cytosol of their body, and then which in turn comes from this intense UV absorption of these proteins. Now, once again, we're actually seeing this kind of interconnection between the absorption of UV, which is a shorter wavelength, and also high refractive index and longer wavelengths. So they're taking advantage of exactly the same physics that you did. The same physics, that's right. I wanted to ask you, it just occurred to me, about the proteins in the eye. So, like, one of the miracles of human biology is how clear our lenses are and our eyes. Is this a similar phenomenon, or is it different?

20:04I'm so glad you're asking this, because we actually also recently looked into this. This is just amazing, you know, to see that You know, we have various, we have probably, I don't know the number exactly, but thousands of different proteins in our body. The crystalline, this is the protein that is found in cornea and the lens, which is important for the transparency in our eye and also the high refractive power for our stocetins, is one of the proteins that actually is enriching the amino acids that give rise to the most intense UV absorption. Oh, wow. If you think about it, we have plenty of different natural amino acids, but actually only a subset of them, very small fraction of them, have the most intense UV absorption.

20:46And then crystalline is one of these proteins that has such a high concentration of its strongly UV-absorbing molecules, such that it derives a very high refractive length. If you compare crystalline with other proteins in our body, crystalline is the highest, has the highest refractive length. And that gives rise to transparency and a refractive power. and and and it preferentially uses these special amino acids these are the amino acids are the building blocks of proteins there's 20 of them as you just said and you said some of them are especially good at this uv manipulation and is it enriched in those kinds of amino acids in crystalline the protein does that okay now i wasn't expecting to go in this direction but that makes me wonder if we could engineer other proteins to be more transparent by making replacements with these kinds of amino acids in areas where it doesn't mess up the function exactly so this is actually a really really really great suggestion so we're actually already looking into this so one of the things we found is that first of all the components the concentration the percentage of this uv-absorbing amino acids is one thing and the other thing is actually interaction of the amino acids it turns out actually when the amino acids actually interact with each other for example in a polypeptide the interaction itself could also enhance the absorb.

22:02And then now really, this really begs the question. What kind of amino acids we should put in? What kind of sequence we should design these amino acids to make the most UV-absorbing proteins such that if you overexpress these proteins in a cell, in a mammalian cell, we might be able to actually make a transgenic and the adherent to be transparent mouse and even make some of the mammalian tissue transparent. My mind went exactly there that if you make these substitutions and they're compatible with life now we have partially or fully transparent mice running around you might not start out with mice right you might start with something a little bit more simple like there are these very small worms that we maybe could do first but okay that's very very okay thank you for ends i'm glad i asked that because that's fascinating and and it turns out that this is a this phenomenon of the uv connection with the visible is the trick that nature has used exactly as you have exactly i wanted to move to one other thing you're working on because it just also sounds similarly magical which is the idea of um light that you introduce into the circulatory system intravascular light sources so just like before why would we need these and then tell me how we might be able to build them or how you are able to build them yeah well actually all this work i actually started from the you know the inspiration i got from uh you know reading a lot of the papers from the Dysports lab.

23:25We got really inspired by all the work, pioneering working optogenetics from the Dysports lab. And then we realized that actually for a lot of the applications involving the delivery of light into the body, such as optogenetics, we have to get our own. So just for people who are not aware, optogenetics is the idea that we can control the function of genes in our bodies or gene products using light. And it was invented by among others, our colleague Carl Diceroth at Stanford. But this has created a revolution in the ability to turn, for example, neurons in the brain on and off using light, but you need to get the light to turn on and turn off at the right parts of the brain.

24:03Exactly. Yeah. So we actually, we found that actually in order to get the light deep in the tissue, especially in the mass brain, we have to overcome the scattering, the opacity of the inherently opaque tissue. Yes. So conventionally, if you want to insert optical fiber, we actually asking the question, can we get rid of the optofibrocyl kind of these invasive to the tissue by using a light source that is traveling inside the body and only gets turned on with a form of energy that can non-invasively penetrate through the tissue. And in order to achieve that, we cannot rely on light itself because we have just a scene of scattering light.

24:41Yes, and we just spent 20 minutes about how hard it is to get light into deep tissue, yes. And then one actually alternative form of energy is actually ultrasound. Is what? Ultrasound. Ultrasound, okay. We know that ultrasound is widely for deep tissue imaging in the hospital. It goes through centimeters of the tissue versus less than a millimeter for light. Yes. But unfortunately, ultrasound itself is not light. These are two very different energy modalities. And the question is, how can we convert ultrasound into light? We actually created technology that allows us to, quote unquote, see the sound, which is basically using a material that upon ultrasound stimulation can release light emission.

25:23And then one of these types of materials is called mechanoluminescent materials. Basically, you create mechanical stress in a material using ultrasound, and then they become luminescent, which is light emission. And the interesting of these materials is that they can be made so small that we can inject them into the circulatory system. and during the circulation they only get activated by the ultrasound wherever and whenever you focus the ultrasound in a particular deep tissue location and only at that location can you get the light emission so this is actually a very simple idea so this special material that you've created circulates everywhere in the body but it's dark so to speak but it can be activated when you do a focused bit of ultrasound that then makes it light up.

26:11And now does it light up to the visual spectra or do you have control of the wavelengths? We have control over the wavelengths. We actually have demonstrated that it can emit various different colors from blue all the way to red. So this is exciting because as I explained with the optogenetics, you might want to turn certain neurons on or off. And then I'm sure people like Carl, our friend, and others would say, what is the resolution? How precise can we focus ultrasounds so that we just turn on the areas with beautiful control of exactly where it is? I just don't know. I see these fuzzy pictures of my grandchildren in ultrasound.

26:51So how good is ultrasound at focusing? Excellent question. It actually depends on the frequency of ultrasound being used. Just like light, the resolution of ultrasound is diffraction limited. So which means if you're going to use, let's say, 1 MHz ultrasound, which is commonly used in our lab, it gets us a resolution of about 1 mm. But if we are able to go up to 5 MHz, that goes down to 200 microns. And 200 microns resolution is actually quite similar to that achievable using any time for fiber. Yes, yes. That's 10 or 20 cells. Yeah. So that's really very precise, and they should be pretty happy with that.

27:28So what is the status of that technology? Is it still experimental? Have you injected it into mice or other living beings? And what kind of results have we seen? We actually are, we have published a few papers on this. And then the latest work that we feel very excited about is actually using this ultrasound to produce light emission at multiple locations in the same mouse brain. So, conventionally, you think about implanting optical fiber. It's actually so invasive that you can only implant in one, maybe maximum, or two or three locations. But now, this is actually with ultrasound. this becomes a quote-unquote maybe virtual light source without a physical implantation.

28:05And then basically you can dynamically target different brain regions, producing light emission in an orchestrated way, almost like playing the piano with 10 fingers instead of like one thing at a time. Right. Thanks to Guo Songhan, that was the future of transparent tissue. Thank you for tuning into this episode. You know, we have more than 250 episodes in the back catalog, and you'll find an amazing array of discussions on the future of anything. Please remember to hit follow in whatever app you're listening to. That'll guarantee that you always get alerted to our new episodes, usually coming out on Friday, and you never miss the future at all.

28:41You can connect with me on many social media platforms, including LinkedIn, Threads, Blue Sky, Mastodon, at R.B. Altman, or at Russ B. Altman on Threads. You can also follow Stanford Engineering at Stanford ENG or at Stanford School of Engineering.