Cole Hons: Hi, this is Cole Hons from The Symbiotic Podcast. This week, we're taking a break from our series of COVID-related stories to bring you an episode we recorded back in February, just before we all went into lockdown. It's a really cool conversation with three brilliant women working with bleeding-edge microscopes at the convergence of materials and life sciences. Their work holds great potential for the advancement of medicine, including promising new diagnostic methodologies for the early detection of breast cancer. I hope you'll enjoy this detour from our pandemic-related coverage.

Intro (Nina Jablonski): Evolution involves more than the survival of the fittest. It's also about the survival of the most cooperative and mutually beneficial relationships are critical to the survival of every species. Welcome to The Symbiotic Podcast, where we will explore the collaborative side of life and work to consciously evolve science itself.

Cole: Greetings, fellow Homo sapiens and welcome to The Symbiotic Podcast! I'm Cole Hons from the Huck Institutes of Life Sciences at Penn State, and today I'm very excited to have three guests here with me, colleagues from Penn State. To my left, we have Deb Kelly, Professor of Biomedical Engineering and Director of the Center for Structural Oncology at Penn State. Her research involves the innovative use of cryoelectron microscopy and in situ transmission electron microscopy to deeply investigate the tiniest of biological structures. Dr. Kelly's mission is to engineer new molecular paradigms to create a world without cancer.

Then we have Jennifer Gray, an assistant research professor and staff scientist at Penn State's Materials Research Institute, specializing in transmission electron microscopy and cryoelectron microscopy. As a highly specialized technologist working at the convergence of materials and life sciences, Gray helps researchers from both camps to benefit from the breakthroughs made by the other.

And finally, we've got Cameron Varano, Assistant Research Professor of Biomedical Engineering at Penn State. Her research in structural biology involves prolific use of cryoelectron microscopy to examine and categorize exosomes involved in the development of metastatic cancer.

Thanks again for being with us today. Let's kick off this conversation talking a little bit about technology, if we could. We're sitting here in the Millennium Science Complex at Penn State, which is a pretty phenomenal building we're going to talk about a little bit today. Later on, we'll talk about your specific research and some visions we have for the future, but if we can start with the technology, for people who don't know, let's go through a little brief history of electron microscopy. Where did it all start?

Jennifer Gray: Well, it really started off, I think, around 1930 with Ruska, and he was one of the key people who developed the technology for electron microscopy. And it was really made possible by coming up with electromagnetic lenses, which is a way you can focus an electron beam. So when we do electron microscopy, we're using focused electron beams, and you need that electromagnetic lens to do that.

Cameron Varano: Yeah, so it was long desired to use electrons to view things because it's all about the wavelength. Size always matters. And so with light, the wave is literally so large that when you want to look at things on the atomic scale, or even looking within the details of a cell, the components that make that up, the wavelength of light is literally so large it just kind of washes over what you're looking at. And so electrons, their wavelength is such that we can look at these things, but you can't use the lenses that we use in an optical microscope to shape these. So really the development of how to shape and form your beam was the first challenge, so they did that and then it progressed to collecting this data on film. And then, in the 1950s, it became more widely used, and then it was cryo-EM, which I think we're going to get into.

That was really in the late '60s, where we started seeing the first freezing of substances, which had a lot of challenges. And that's why three people won the Nobel Prize for cryo-EM, to combat those three specific challenges, which is, how do you put the sample in the microscope and maintain it in that vacuum? And the second challenge being, how do you then interpret that data? And then, how do you use that to evaluate biological mechanisms? And so the freezing mechanism was really... Actually, it was first done by Bob Glaeser and Ken Taylor, but really streamlined by Jacques Dubochet, and so he was one of the winners, and then Joachim Frank and Richard Henderson, respectively, did the calculations and the first atomic reconstruction.

So that's sort of where it progressed, but it kind of stalled until, I would say, the late '80s, early '90s, when we started trying to digitize things. By "we," that's not me, but the community. And so then we started moving to digitation. Is that a word? I don't know. Digitizing things, yeah. And so that's where we saw the advent of CCD cameras, and then finally, we've sort of had a boom now because this convergence happened with cryo-EM, and so I think everybody in the EM field has benefited from that. But because of that boom, we've seen a lot more advances in our data interpretation and in the way we collect our data. So that's a brief history.

Cole: Thank you! I didn't realize it went back that far, because everything I hear is that recently, suddenly we can see things that are so much smaller than we ever thought was even possible. I've had high-level professors tell me, "When I was in school, they said this stuff was impossible." And I always had the sense it's in the last 10-15 years, but this stuff's been coming for a long, long time. Was there something-

Deb Kelly: Yeah. There was a revolution in the cameras, the direct detectors that enabled this more high-resolution revolution that's taken over the cryo-EM field. And each of these modern cryoelectron microscopes are so powerful. It's 10 times the power of the entire Boston subway system per microscope that you're shooting at these entities all at once! And they are capturing all these atomic events that you couldn't otherwise see. So the atomic events are there to be captured, but unless you had a really good camera... Every year when you get a new iPhone, their camera improves. The same thing has happened in the cryo-EM field, and now these new detectors allow you to capture this atomic-level event in pristine detail, and that really pushed everything in the last, maybe, five to 10 years or so as they came about, and moving the field forward. So every time computational routines improve, also all sensing technology improves, so pushing computers, getting high-level, high-performance computing, really allows us to see in exquisite detail the full, unbridled power of these electron microscopes, many of which are contained here in the basement of MRI and the Huck Life Sciences Institute.

And when I first visited here, I was so duly impressed with the convergence between all the instruments. I said, "Oh, what a cool playground. Let's add more to it and just keep growing." And we've been really lucky to work with Jen and Clive's staff in MRI, as well as that of Huck Life Sciences, so I think the possibilities are endless the way the field is growing, as well as the instrumentation here at Penn State.

Jennifer: And there's more you can do with the electron microscope as well, other than just taking images. You can collect spectrum that will tell you what atoms are present in the material, and different types of spectra that can tell you the chemistry and the bonding, so you can get diffraction patterns, so I always say that TEM is one-stop shopping. You can pretty much figure out everything about your material as long as you can make a sample, which is pretty key.

Deb: Yeah that's really [inaudible crosstalk] challenge is how to make a sample.

Jennifer: Right. Your sample needs to be electron beam-transparent.

Cole: Electron beam-transparent.

Jennifer: So that electron beam has to be able to go through the sample.

Cole: I see. Which means super, super thin.

Jennifer: Which has to be very thin, depending on what elements are present in your sample. If you have a gold sample, it's going to need to be a lot thinner than a carbon sample or a biological sample, which can be thicker because it doesn't scatter electrons as much.

Cole: Interesting. So for our listeners and our viewers, I want to talk about this term "convergence." Now, we all know what convergence is. We're in this building. We're in a building that was created for convergence. But does anybody want to tackle, just to break down or break out what that means, in case people aren't familiar? Earlier, Cameron, you said convergence revolutionized things in a big way.

Deb: Yeah, so historically, getting back to what Cameron and Jen were talking on, we had the use of electron microscopy for medical pathology versus materials science, and it was only in maybe the '80s or so where these two fields grew together and said, "Hey, we can learn from each other." And now, cryo-EM was growing and taking routines used in materials science to make better specimen, and computational routines from cryo-EM are now being used in materials science, and as the two fields grow in parallel, they also need to cross over and grow together. And so I think we're really good at doing that here, so then we're not just looking at thin sections here, medical diagnoses or metal fractures in materials. We're actually looking at mechanisms and seeing things as they would naturally occur, and I think a good way in which convergence is typified is in this new field of in situ electron microscopy.

And so what that is, is it allows you to record things using these advanced detectors in real time, as they actually occur. You can do this in biology or in materials science, so Jen and I just got back from a conference in which people were looking at both sides of things, hard and soft polymers, to try and understand things. And so by "polymers," we mean lots of different materials, whether they're made up of soft atoms or hard atoms. And really, convergence is working across disciplines and across fields to reach common questions and look at things related to mechanism: how things are occurring, what does it mean, how is it in the context of the scientific questions you want to answer. And seeing is believing, right? So when you start to see these things as they naturally appear and occur, no matter what the discipline is, you're really learning more about the questions you have in mind to answer mechanism.

So to me, convergence is answering the right questions at the right time, related to a process you wouldn't otherwise see. And imaging is excellent for doing that.

Cole: Right on, thank you.

Jennifer: And also, we're trying to really do cross-fertilization of different electron microscopy techniques down here, so in the cryo-EM world, they've really pushed cryo-EM and capturing images at very low doses with these new detectors, and that's something that hadn't really been applied to materials science so much before. So we're taking those techniques that were developed so strongly for cryo-EM and now applying them to systems in materials science that really damage quickly under the beam, so we're stealing from them and applying them to new materials systems that are very beam-sensitive.

Cole: Well, that's exactly what our podcast is about, people learning from one another and tricks of the trade.

Jennifer: And on the other side...

Deb: It's very exciting.

Jennifer: Yeah.

Deb: It's an exciting new way to think about seeing things differently.

Jennifer: Yeah, and on the other side, we can take these materials science methods, like the spectroscopies, like scanning transmission electron microscopy, and apply them to biological samples, which hasn't been done much before.

Cole: Fantastic. Well, also for our listeners, we're talking about TEM and cryo-EM, so maybe we can break that out a little bit too and let people know what the difference is. Cryo's about freezing, correct? Is that-

Cameron: Yeah, so one is really just building on the other, so TEM, as Jen, I think, said earlier, is transmission electron microscopy and cryo-EM is cryogenic electron microscopy. The T in TEM stands for where we're detecting the electrons, so they're coming through the transmitted one. And when we're doing cryo-EM, we're often using that TEM, so they're really one and the same T. I guess cryo is a subcategory of TEM. [crosstalk]

Cole: Like every square is a rectangle, but not every rectangle is a square.

Cameron: Yeah, so you don't have to-

Deb: It's cryo-TEM, in fact. People just leave off the T.

Cole: Oh, okay, right on, right.

Cameron: So yeah, it just means you're not freezing your sample, and there's other ways to encapsulate your sample.

Jennifer: Yeah, and it's liquid nitrogen temperatures, is what we're using.

Cole: For cryo.

Jennifer: For cryo.

Cole: Is it-

Jennifer: But regular TEM is just done at room temperature, whereas for cryo-TEM, you're using special holders or special liquid nitrogen cryoboxes to keep your sample frozen.

Cole: A lot more expensive, I imagine, in cryo versus-

Jennifer: Yeah, it adds some cost [crosstalk].

Deb: Tedious. Tedious and more finicky. Everything has to maintain this pristine -180 degrees Celsius or colder the entire time. It can't warm up at all. It has to be transferred into the electron microscope column quite quickly, as well, whereas in TEM, you can do that at room temperature if you need to. It's not as difficult to just put something in dry or at room temperature, as compared to frozen hydrated, which is what people are typically doing in cryo-EM, especially with biological samples.

Jennifer: Yeah, there's a difference between just cooling a dry sample down versus freezing a wet sample and transferring it into the microscope, and that's way more difficult than just taking a dry sample and cooling it down. That's very easy, but when you have something that's in the liquid state, and you have to freeze it and get it into the microscope without getting a bunch of ice build-up, that's an art.

Cameron: But coming back to convergence, freezing has been a challenge, it's said, but we really utilized some of the advances in materials science to help allow us to be more specific in the way that we freeze. I often say that in the past, from the invention of vitrification until even currently, we've relied often on a sort of Goldilocks method when we're freezing. So when we freeze something, we know that a certain area's going to be too thick, and we know a certain percentage is going to be too thin, but we're hoping this one area will be just right. And we don't exactly know where that area is, so we're doing it on a gradient.

But if you were able to build a material that would allow you to then mediate that process, which is not something that biologists have thought about in the past... Things that we typically interact with would quite literally burn up or be ineffective in that system, so in materials science, we've seen a lot of advances with those materials, and so those materials now have transitioned. And while people in materials may be borrowing the methodology, they are consequently also adapting and helping us change that methodology to be more predictable, so that it is less expensive.

Cole: Cool.

Cameron: Yeah.

Deb: And the development of new materials is totally changing the way you can prepare samples as well. So one feeds into the other, obviously.

Cole: Yeah, so [crosstalk].

Jennifer: Nanofabrication.

Deb: Yeah, we're got fantastic nanofab here.

Cole: That's right. I got to go tour through there. I was in the orange room, wearing my little outfit and everything. There was a journalist there who passed out, actually. She was overwhelmed. We caught her. Just as an aside, in the middle of the tour, for the journalist, we caught her and wheeled her out of there. Something about being in that environment, the clean room, was a little intense about 15 minutes it, but it was amazing to see what goes on there, just atoms and the guy with Quality Control having to send stuff back again blew my mind. Right here in this building, yeah, it's pretty cool stuff.

Speaking of the building, I've heard it said that we have the best building in the world for these microscopes. Does anybody want to back that up with some factoids or some perspective on that?

Jennifer: Yeah, there's been some serious engineering that's gone into this building. The microscopes are actually in an underground bunker, I call it, which sits underneath the garden, so it's a separate building that's underground that was built primarily for the electron microscopes. And the garden is over top so that there's no cars driving over it, or bicycles riding it, or anything that would cause vibration, because when you're trying to look at something at the length scales that we're looking at, if you have any kind of vibration or anything, that's going to destroy your image. So they're called the Quiet Labs, and again, a lot of engineering went into that. There's huge concrete pads that sit underneath each of the microscope rooms. Each of the microscope rooms is its own little building within the bunker. There's special cooling panels on the walls, special airflow socks on the ceilings, so a huge amount of engineering went into this building.

Deb: Yeah, it's pretty phenomenal, the engineering that had to be well-thought out in advance. So then, as you add more instrumentation, everything has to be duplicated and replicated up to standard, so you can actually see these, we're talking about imaging at the atomic scale, so any vibration you're going to see is going to dampen the information you're gathering. So the exquisite design of each of these little bunkers, with decoupled floors and vibration-free platforms that they're sitting on, it is really extraordinary to see.

Cole: Yeah, I saw an image that was taken by one of these incredible, powerful microscopes before it came in this building-

Deb: Before and after, yeah.

Cole: And it showed what it could do, and then they brought it in-

Jennifer: The AFL one, yeah.

Cole: And the same instrument had a completely different resolution just based on where it was located.

Deb: It's pretty phenomenal.

Jennifer: The other benefit of those too is, before they installed one of these instruments, the company that manufactures the microscope will come in and do site surveys, testing to make sure the rooms meet their specs, because they know if the room doesn't meet the specs, then they won't be able to meet the microscope specs. And when they come in here to install a new microscope, they always know that we're going to be way below specs for things like vibration and noise. And because of that, the actual microscope specs, we always beat them as well, so we get even better resolution from our microscopes than what's specified by the manufacturer.

Cole: So the manufacturers must love us, because we're really using their stuff in the best way possible.

Deb: It's an ideal environment, absolutely.

Jennifer: Yes, it is. They don't have to worry about something failing before they do the install.

Cameron: And beyond that, MRI in Huck is professionally staffed. I cannot express how rare that is. I don't know the numbers, but I imagine it's one of the few where it is professionally staffed. And I think that makes the difference in the training on all levels, and on the troubleshooting, and on the way that you think about things. I certainly get a much broader perspective on the opportunities that we have for analyzing and providing more rigorous data. When I'm having a conversation with Jen or any of the other staff who are down there, thinking about every day and seeing other samples that are being imaged, so they really are cross-pollinators on a very analytical... And having professional, highly-skilled staff is just... The building is phenomenal, but the building would not function without those professional pollinators.

Deb: Yeah, professional researchers like Jen, for instance.

Jennifer: Yeah, they made a conscious decision to really invest in staffing in the labs downstairs, so there's dedicated Ph.D staff for all the different areas in the basement of characterization. That's unusual.

Cameron: It is unusual. There's an article on this. I was down in Hershey for a conference last week, and they noted how important cross-disciplinary research is for us moving forward. But this article had evaluated different sites, where interdisciplinary research was supposed to be taking place and was it effective and what was going on. And the article was mostly quite critical as to the inability to effectively do interdisciplinary research. There was one example, and it was Penn State, and one of the reasons was because it's professionally staffed. And that makes a huge difference, and that the staff is on all levels. So those who have their Ph.D and are down there working with your sample, and those who are above and orchestrating things are the experts in their field. So you're pulling out somebody who is highlighted and lauded by their colleagues to then help facilitate this, and that's very unique.

Cole: Thanks for pointing that out, because I'm a staff person at the Huck. I'm not a scientist. I work on the communications side, but I work with all the folks in the 11 different core facilities across the Huck, and it is very unique. And as I've done my due diligence as a communicator, I go, "Wow, there's not too many other people in this game the way that we are, and I think we are really doing this the right way," so thanks, Jennifer, and all your colleagues across all these very open... It's a very open kind of an approach that we're taking at Penn State that just opens up to the entire community, "Come use these facilities, learn, cross-pollinate the ideas across the disciplines so that we can collectively put our heads together and really make some advances and do things in a new way." So it's exciting to hear more details about that.

I'm Cole Hons. This is The Symbiotic Podcast. We're going to take a little break and come back with part two to start to talk about the research that we're doing in these phenomenal facilities. Stick around. Thank you.

Commercial: The Huck Institutes of the Life Sciences at Penn State offer six inter-college graduate degree programs in bioinformatics and genomics, ecology, integrative and biomedical physiology, molecular, cellular, and integrative biosciences, neuroscience, and plant biology. We also offer an accelerated professional science master's program in biotechnology. At the Huck, we immerse students in a groundbreaking environment built on interdisciplinary collaboration among some of the world's most innovative research scientists, and our students receive unparalleled access to bleeding-edge technology in world-class core facilities. If you're looking for a deeper, more holistic grad school experience, you owe it to yourself to look at the Huck. Visit us online today at huck.psu.edu.

Cole: Hi, welcome back to part two of The Symbiotic Podcast. I'm here with my colleagues from Penn State, talking about using incredible cryoelectron microscopy to look at teeny-tiny little parts of life to solve really serious problems, and we're about to talk about that research. Before we get into the specifics, and you can actually speak about the specific research you're doing, but what are some of the major hurdles at working at such a tiny, tiny scale? Now that we can see down to that scale, what are the biggest hurdles?

Deb: I think it always comes down to preparing a perfect sample, or as near to a perfect sample as you can. It doesn't matter how good the microscope is if what you're sticking into it to observe is a big mess. So you really have to be very fastidious in preparing your sample the right way. Be it in materials or in biology, it comes down to sample prep, and that's one of the big pushes in the next revolution in cryo-EM nowadays, is coming up with new ways to get reproducible, better samples all the time, and not waste people's time and be more efficient at it.

Our lab has done a lot in sample preparation over the years. I started in that area back during my post-doc, and I've translated that forward all the way throughout my career. And I'd like to say that we prepare samples the right way, natively from cancer cells, and that goes into a bit about our research in that we have the ability to use different types of materials to prepare these very pristine samples directly from cancer cells, and then look and see what's wrong with these entities. And it's just something we can do about it to make them better. It's a big issue in materials as well, right, Jen?

Jennifer: Oh, yeah. Sample prep is key. If you put crappy samples in, you're going to get crappy data out.

Deb: Yeah. Or no data.

Jennifer: Or no data.

Cameron: It's really a mindset. I feel like you have to just abandon the idea of time and be like, "It doesn't matter what else is going on."

Deb: Yeah. Throw your phone away. Do what it takes.

Cameron: "If this takes me two hours, my sole focus needs to be here in this moment, and slowly going step by step." So it's very antithetical to the rest of the day, so you have to take a deep breath in, exhale out, and then focus. And I think that's true across-

Jennifer: And you have to have a lot of patience and again, you have to be able to focus and do this really tedious work to make these tiny samples, and it's not for everyone. I think it takes a certain type of personality-

Deb: Yeah, a certain obsessive skillset-

Jennifer: To be able to do this.

Deb: Of a surgeon or a jeweler, or someone at that level.

Jennifer: Exactly, yeah.

Cole: It reminds me of art. They say, "You can't rush great art," [crosstalk].

Jennifer: Right.

Deb: Or music. That OCD stuff of doing it over and over and over again until you get it right.

Cole: Real art form preparing the samples. Is it more difficult to work with biological samples than materials? Or are they both difficult in their own unique way?

Jennifer: I think they have their own different-

Deb: Unique difficulties, yeah.

Jennifer: Issues, yeah.

Deb: Because soft materials can be just as challenging as biology, if not harder. I think sometimes hard materials, you can't take it for granted that you're going to get a good image out of them, but then some of the analysis ends up being harder, right? So they're all uniquely difficult. It's not for the faint of heart. You just have to be very persistent, just like anything else in science or academics. Persistence and attention to detail wins the day.

Jennifer: And a little bit of trial and error.

Deb: And a little bit of luck.

Jennifer: Yeah, and luck. A lot of luck.

Cameron: A lot of-

Jennifer: Serendipity.

Cameron: Yeah, I think a lot of luck. But I also think just a general appreciation for each step.

Deb: Mm-hmm (affirmative), absolutely, yep.

Cameron: Because otherwise, you will want to rush through it. But "Okay. If I'm thoughtful and careful in this moment, I understand the implications." So perhaps that's delay discounting, but-

Jennifer: And that can be hard to teach people. It's difficult.

Deb: Yeah, it's a certain personality type it selects for as well.

Cole: So you have to have some innate ability to have game in this sample preparation field.

Jennifer: Right, or else it's just going to take you so much longer to be able to do it.

Cole: So do some scientists go down that route and realize they don't have a knack for it and have to shift gears into another aspect of the work? Does that ever happen?

Deb: That's when they go in to-

Cole: Or a data analysis route?

Deb: To come and nag Jen, generally. Or Carol, Carol Bator.

Jennifer: Yeah. They'll say, "Can you get my data for me?"

Deb: "Can you help me?"

Jennifer: And then they can analyze it.

Deb: And Jen's like, "I can, but it's going to cost you."

Cole: Back to what Cameron-

Jennifer: I'm not cheap.

Cole: Yeah, right. That's what Cameron said earlier about staffing.

Deb: You get that high level of professionalism.

Jennifer: Right, exactly.

Cole: Staffing the facilities with people who know what they're doing.

Cameron: Yeah, because we certainly all can... We don't have enough time to be experts at everything, right? So if you have experts, and you have people who can be on board, and even if perhaps, they have the personality, is that the best use of their time? No, if they have a grant, perhaps their best use is collaborating with Jen or someone else.

Jennifer: Right. But also, some of the really advanced data reconstruction computer methods, that's something I don't have a lot of experience in, and it would take me way too long to be able to learn that, so I kind of stick to what I'm good at and leave some of that other stuff to other people.

Deb: Yeah, it all starts with a good sample. And from there, you can get good data, and from there, you can get good analysis or computation, so it really is all about the care you put into preparing it, like a good meal, if you will.

Cole: Got it. So once you have that good sample, what are you working on now? What samples are you working on? And let's talk about the research a little bit.

Deb: Well, we work in a few different areas, primarily related to breast cancer and the molecular culprits that help cancer cells stay alive, and how we can shut them down properly. So there are a lot of genes within our cells, and some of those genes are built to help protect cells from damage and things that go wrong. And when there are problems in those protective mechanisms, then disease can arise, and so we look very deeply inside these cancer cells and pluck out the proteins of interest and harvest them and say, "Why do they look different in cancer cells, and what can we do to either correct them or shut the cell down entirely so that we can create new paradigms and opportunities to look at cancer differently and look at new therapeutics in a way that hasn't been realized before?"

And we do this in a few different proteins in general, and in a few different genes. We work on the breast cancer susceptibility protein, also known as B-R-C-A-1 or BRCA1, and our new area of investigation is in this tumor suppressor gene, meaning a protein or gene product that works naturally within the cell's defenses to prevent cancer from happening, and that's p53, and that's another popular tumor suppressor we work on. So we work on these mechanisms to try and understand what's going wrong. Why are they no longer protecting cells from the formation of cancer, in particular breast cancer? And then those are going over into other types of cancer systems that we're interested in, because they're found in every cell. So that's are main focus in the Kelly Lab, is understanding cancer differently and how can we correct it or wipe cancer out or better manage it at the molecular level.

Cole: So what would a sample look like to give you the information and the data that you're looking for?

Deb: Little blobby proteins. We look at little blobby proteins, and then we do a lot of processing of these pictures. Think of pointillism in-

Cole: Pointillism.

Deb: Think of pointillism, when you're looking at a picture made in an area. You get these little point and density spots in space. But then you put all these little points together, and you come up with what they look like in 3-D. And those little points are like the atoms that make up the proteins, and then what's the blueprint of how they're put together? And then we use computers to try and define that, and come up with a better three-dimensional overall architecture of what these proteins look like. And then we see inside these proteins little crevasses, or little places in which you can design drug pockets, or things that you can do mechanistically to correct them when something does look wrong, or how can we look at them differently? So we look very deeply down at these little blobs that contain atomic information, and we use high-performance computing to come up with what they look like in reality, in 3-D, as opportunities for better treatments and therapeutics.

Cameron: If I can jump in with an analogy. So if you were to turn off the lights, and then shine a flashlight behind your hand, and your hand up against the wall, you would see a shadow of your hand, right? That's sort of what transmission electron microscopy is, so we're looking at what transmits through. And so what we see is the outline of your hand, and then we try to find that hand in multiple different orientations, so then computationally, you can take those different orientations and those different bends to then reconstruct it back, and then you can say, "Oh, she has thicker fingers that are kind of short," or "There's a bigger palm, and so that's why that person is better at catching a baseball."

So first, we start with the electron beam shining. It's giving you these 2-D images, so that's really where the sample preparation comes in. You really only want hands, then. You don't want aberrant feet showing up, because that's something that's going to make it look like you have really short fingers and a giant palm, and you'll lose your thumb. So then, in a disease, the way this looks like, if you had a mutated hand, and so you don't have a thumb, then we're able to look at those and see a hand without a thumb, and realize that grasping something is much more challenging. And so then that provides insight into, "Well, okay. What we see is this missing thumb, so is there a way that we can provide a therapeutic that functions as a thumb with this hand, so that we're able to..." But it's on the protein level.

Cole: I was about to say [crosstalk]-

Cameron: Protein level.

Cole: The hand is a protein in this thing.

Cameron: The hand is the protein in this case.

Cole: I see the BRCA1 protein [crosstalk] hand.

Cameron: Yeah, and-

Deb: Cole, have you ever broken a bone in your body?

Cole: Luckily, I've avoided it somehow.

Deb: Have your kids or wife or anyone you know in the history of your life ever broken a bone?

Cole: Yeah, I'd say I've had people... Yeah, sure, sure, in my life.

Deb: And you've seen an x-ray, where there's a break?

Cole: Yeah, sure, yes, uh-huh (affirmative).

Deb: Well, in that x-ray, you see there's a break, and you put a cast on it, and then you see it healed up?

Cole: Yes.

Deb: That's what we're doing on the molecular level.

Cole: Wow.

Deb: There's a break? Let's fix it.

Cole: Yeah.

Cameron: Yeah.

Cole: That's fantastic. So that beam, the electrons are shining that flashlight from all these different angles.

Deb: Think of it as the x-ray view.

Cameron: Yeah, so you're-

Cole: And then you put it together and you see-

Deb: Like a 3-D x-ray view of a broken bone.

Cameron: Yeah.

Deb: [crosstalk] fix it-

Cole: And that is why your center's called Structural Oncology, the Center for... Because it's all about the structure.

Deb: Because [crosstalk] these minute structures and how we can fix them in cancer.

Cameron: And so that's really important. It'd be like blindfolding somebody and then saying, "Okay, go mow your lawn." That would be really challenging to navigate that.

Deb: That'd be really fun to do for someone, actually.

Cameron: You wouldn't know where to go. You may end up mowing your neighbor's lawn because you're like, "I'm not sure where my grass ends," and you're banging into things.

Deb: That's fun.

Cameron: Solving a problem without having visual feedback is not something, as humans, we are equipped to do.

Cole: Got it.

Cameron: We really need visual... That is one of our strongest senses, particularly for problem solving, so being in the electron microscope is sort of like, I don't know, being able to go into the Magic School Bus and go down onto the level of these things and get a visualization of what's going wrong, and having a very pragmatic way to go about solving-

Deb: And I bet if you did mow your lawn blindfolded, you might break a bone there, Cole.

Cole: Yeah, it would suck. Yeah.

Deb: [crosstalk] if you want that experience.

Cole: I might have a mad, mad moment of rolling over [crosstalk].

Deb: Me too, yeah, so-

Cameron: Yeah, but it would be an absurd idea, right? Trying to solve problems without being able to see them. Not something we want to do.

Cole: Got it. So we're able to see these things, now, and when you collaborate, I see Jen's helping you prepare samples, yes?

Jennifer: Not really, because the type of sample prep they do is very different than what the materials people do.

Cole: Oh.

Deb: We collaborate in other ways, like on other projects, particularly with this new liquid phase imaging that we're doing. Pretend you have a cryo-EM sample and it's frozen. Okay, now melt it. Oh, and then you see things move, because it's contained in liquid. And you put in the microscope and you watch it move and do its job. We're collaborating in those areas, with Jen and the MRI team rather than with the fixed frozen items that we look at.

Cameron: One of the things that I've worked with Jen and talked about with Jen is, when we're making these new sample prep platforms. People have very valid questions about what does that look like, and Jen has the expertise in these different forms of microscopy for analysis to be like, "Okay, how do I measure ice thickness?"

Jennifer: Yeah, that's something you can do with spectroscopy, is take measurements of thickness and things like that. So again, it's my background in the materials science analytical side that I can bring to the table to help them in new ways.

Cole: Cool. Does some of this inform some of the custom technology I've heard you talk about? I'm looking in my notes here that it's the affinity capture platform, not the cryo-EM-on-a-chip, it's the affinity capture platform. You said it was like Velcro-

Deb: Oh, you could use both.

Cole: Oh, both operate kind of like Velcro?

Deb: Yeah, you could use both. Jen was using some of our chips, this cryo-EM-on-a-chip technique, to analyze some materials, and I think it crossed over nicely, depending on the sample prep.

Jennifer: Right. And also, when you have samples in liquid and they're moving around a lot, it can be difficult to analyze them, so even from a materials perspective of "We have polymers or something, and we're trying to get an elemental map." If they're moving all over the place, we can't get that, and so being able to somehow attach them to the windows in the chip would be really helpful. So that's how we can take this technology developed for cryo-EM, or not cryo-EM, liquid-

Deb: Yeah, or cryo-EM.

Jennifer: Or cryo, and apply it in new ways to materials systems that we wouldn't have thought about before.

Cole: Cool. Once again, we're sharing things about [crosstalk].

Jennifer: Yeah, it's the convergence.

Cole: Terrific. Well, I want to return to the cancer research a little bit, and BRCA1, and just dig in a little deeper on what you do when you're gathering all these different shapes that we were talking about of the proteins, the missing thumbs and all this. Where are you in your research with that? How much data have you collected? And what does that all look like right now?

Deb: Sure, so there are different mutations in the BRCA1 gene that translate into the gene product, which is the protein. And so the general shape of the protein looks like the letter C, if you will. And there are certain mutations that cause the cell to react in a very bad way to the mutated form of the protein. It reads it as mutated, and it says, "Oh, that shouldn't look like that. I'm going to destroy it." And so what the cell naturally does is, it orchestrates a system where tags are added, like a death tag is added to the BRCA1 protein to kill the protein and cause it to be destroyed.

So what you can do if you see this tag added to the BRCA1 protein is, put in an enzyme that clips it off like scissors and just removes it, and then you can destroy its ability to work again in biochemical experiments or in cells. So it's a way that if you are able to use EM, which we've done, to identify, "Oh, that's a problem. Let's fix it," we can enzymatically, or use chemical reactions, to remove these tags so that you can give BRCA1 new life and make it more viable again. So it might be a preventative measure that we can use in patients early on if we know someone has a propensity to mutation that the cell's going to react to negatively. "Oh, well, we'll make sure that that chemical component that can remove these bad tags is always present to keep the protein in its normal functioning form."

So that's a long explanation to say such that if we notice there's something wrong with it, we know how to correct it as well, when it comes to certain of these more toxic BRCA1 mutations. So that opens a doorway to early prevention, not just prevention, but early on. And that's a new way of thinking about potential therapies that could extend life, or have a better management plan for cancer. So the whole world bought into fixing HIV. Not that it's fixed, but people with HIV now can live longer lifespans and healthier lives. So if we could just better manage cancer in the same way, with the buy-in... There's a lot of research being done in cancer, lots of money being thrown at it. But would you believe that with all the money and resources being thrown at cancer, some of these proteins have never been seen before! You want to see who's wreaking havoc inside cells. What does it look like? What can we learn about it? What can we do to fix it?

So that's our vantage point, is, we're going to use these beautiful high-end instruments to see these cancer molecules differently, and then work to correct them or prevent things from happening and have better molecular management plans for patients in place. Really sets a long-term goal for what we're aiming for.

Cole: Early detection and intervention.

Cameron: It's really key, because we have a lot of information. People are signing up and getting their genomic information back, and people are finding out that they have BRCA mutations. But BRCA mutations, while they may predispose you to an increased risk in cancer, don't always result in cancer.

Deb: Yeah, it's not the full story. It's not the complete narrative.

Cameron: So if you're not getting this information, and it's... That's a 2-D information. That's a readout of your code. But if you're not seeing the product, then you're not ensure... Is that protein? It might be functional, or perhaps we need to monitor to the point where it stops becoming functional. And when it stops becoming functional, what does that actually look like? Deb talked about these little extra tags that get put on there. Your genomic information does not tell you about that tag.

Deb: No, you have to look deeply inside the cell and see what's going on.

Cameron: You have to be able to see it.

Deb: And the mutations might say, "Yeah, about 50 to 60% higher chance of getting cancer," but these tags, they, "Oh, that's definitely going to be bad." So is it a way that we could more stratify the disease?

Cameron: Or, more importantly, if you're 23.

Deb: The potential's there to understand who's really going to get it or not.

Cameron: Yeah.

Cole: Wow.

Cameron: If you're 23 and you're finding out you have a BRCA mutation, do you need to take drastic measures? Or do you not? That's varied. It's as important for the patient who needs to take drastic measures as for the patient who does not.

Deb: Yeah, having that information at hand in a way that you would definitely know something one way or another is much more valuable for women to not mutilate their bodies or face the fact of not seeing their children mature and grow up. And if we could have an impact in that area to stratify the disease, we've talked about this for years... And these silly little tagging systems may be the key to why some forms of BRCA1 are more toxic than others inside cells. We're still investigating that area, but it's an exciting new way of looking at the proteins that was informed by imaging and looking at their images, and their three-dimensional architectures and how it all came in to be. And then we biochemically followed up. We did all our cell biology studies to complement our structural findings, and we're really hot on the trail of figuring this out, I think, in a unique way, informed by imaging.

Cole: That's fantastic. Well, it sounds like a huge piece of this is going to be access to these images. If it's deployed as an early-intervention, as an early-detection strategy, then we want to make sure there's universal access for when they-

Deb: Some sort of imaging diagnostics, definitely, globally would be fantastic.

Cole: Imaging diagnostics to make that available to people.

Deb: Or some biochemical readout that we could quickly do in the field, or on a cell phone, for instance.

Cole: Oh, wow.

Deb: We should grow in that area.

Cole: Part three, when we talk about the future!

Deb: Yeah, we should grow in that area in the future. My dream come true, cell phone detection.

Cameron: I think it's imperative and it's also insightful, right? We've sequenced the human genome, so if it was just mishaps in the genome that were resulting in the development of cancer, if it was on that level, then we would have all the information we needed, but we don't. And so it has to be looked at in this more complex, nuanced manner, so you really need to see, "Okay, if you don't have a thumb, you're not able to catch a baseball. But maybe if you don't have a ring finger, you're not as good, but you still can catch a baseball."

Cole: Got it.

Cameron: So what do those specifics look like so that we understand what's going on?

Cole: And that's what you're tackling right now. You're sort of categorizing the [crosstalk]-

Deb: At a thumb hack.

Cameron: I know, yeah!

Deb: Prosthetic thumbs.

Cole: And putting that whole thing together.

Cameron: Yeah.

Cole: You always want to say when will you definitively be able to know what all the factors are to be able to make a real diagnostic. People always give the five-to-10-year, that thing.

Deb: Yeah, I'd say-

Cole: So how far along do you think you are with categorizing and sussing out-

Deb: A time frame of maybe next week to never. Right? I mean, to be broad about it.

Jennifer: Somewhere in there.

Cole: Yeah, right.

Deb: But I think if you... Because she thinks differently, of a different chance of attacking the problem. And so that's why imaging fascinates us. You have the [inaudible] of "Wow, it really looks like that? Oh, wait, what's wrong there? That doesn't look right. What can we do about it? Sprinkle something on it and let's see how we can fix it."

Cameron: We're here on the podcast to say that seeing is the most important sense.

Deb: Yeah.

Cole: Yeah.

Deb: Strangely, this podcast is being visually taped, so we're on board with that.

Cole: Right. Well, thanks so much. I'm Cole Hons. This is The Symbiotic Podcast. We're going to take a little break and be back with part three, where we talk about visions for the future. Stick around. Thanks very much.

Commercial: Hey, Symbiotic Podcast listeners. My name is Jenna Spinelli and I'm part of the team that produces Democracy Works, a podcast from the McCourtney Institute for Democracy at Penn State and WPSU that examines what it means to live in a democracy. Each week, we present intelligent and thought-provoking conversations about topics ranging from immigration to impeachment, and conspiracy theories to climate change. You can find Democracy Works at democracyworkspodcast.com or by searching "Democracy Works" in any podcast app. Thank you to Cole and the Symbiotic team for letting us put this ad in their show, and we hope you'll check out Democracy Works.

Cole: Welcome back to The Symbiotic Podcast. I'm Cole Hons, your host, here with three of my colleagues from Penn State, and we are talking about the use of incredible microscopes to see teeny-tiny, teeny little things that give us new insight into all sorts of problems, including cancer. And for this third part of our conversation, we're going to be looking to the future. And Deb, I know you're a futurist by nature.

Deb: Absolutely.

Cole: So why don't you tell us a little bit... What kind of synergies do you see coming down the pike in terms of, say, materials and biomedical innovations?

Deb: Right, so I love cryo-EM and that's really our bread and butter, but we also work at a different area of imaging called liquid-cell, or liquid-phase electron microscopy, and I really feel like part of the research we do, a major component is going to dive more deeply into that area. And so what that allows us to do is, say we have a frozen sample of something biological, but then it melts, and then the biological thing is alive inside the liquid. That allows us to see this biology while it's in liquid and performing some sort of task, in a quick enough timeframe that we can capture information about it before it burns up in the electron beam, for instance. So I really think the future of what we're going to be doing is analyzing biology in liquid in real time, and also bringing in 3-D insights to do that, so we could look at something in 3-D in real time as it occurs.

So that's almost like taking it to the fourth dimension, looking at changes in 3-D over time. We've started to do that a bit with different types of viruses, and we're working with Susan Hafenstein's lab to look at some of her virus samples as a testbed to try and understand how these viruses look and act and breathe a bit while they're contained in liquid. And then we want to apply those to other samples. Things binding to DNA: do they bind? Do they grasp? Do they twist DNA? What's going on there? Can we look at these things and try and uncover new insights about them? And we're also continuing our work in developing new materials, whereas we can harvest and pluck out these proteins from cancer cells and see them differently, and we're constantly evolving that sort of molecular Velcro technique that we're saving this for in our lab.

So I really think the future's in a few different directions: looking at cancer proteins and other forms of cancer, and how are they wreaking havoc on other forms? Can we be more proactive in prevention or management strategies, as well as can we now melt these samples and look at them and observe their structures and their dynamics all at the same time inside the electron microscope with the liquid-phase imaging? So there are a few different future-thinking areas that we're diving into right now.

Cole: Wow, that reminds me of an MRI, like putting a person or an animal in an MRI and seeing for neuroscience, for example, but being able to do that on an incredibly small scale.

Deb: Yeah. They'll do that at the level of molecules, as well as watch it come to life.

Cole: Wow. What timeframe would you be able to look... You say you're already starting to do this a little bit. Are these nanoseconds or how long-

Deb: Definitely in the millisecond timeframe, for sure.

Cole: Millisecond.

Deb: Yeah, [inaudible] changes that you can see in liquid. Yeah, at least 40 frames per second right now with these new cameras that we have in our new microscope.

Cole: So you're saying biological material stays alive, essentially, before it gets burned up by electrons, essentially, is what you're-

Deb: When you don't the electrons very heavily on it, you just lightly sprinkle them on it.

Jennifer: You got to keep really low doses of a few electrons per angstrom squared per second.

Deb: And we were using one electron per angstrom squared per second over 40 frames, over 20 seconds, so mathematically, very, very little what you would do in just one imaging cryo-EM for the entire image series. So you're dispersing that energy over a larger time frame with a different level of frequency, so you can lightly sprinkle the electrons on and see the images much faster with the way they're integrated in these new cameras and detectors.

Cole: Wow, that's fantastic. What other trends do you see in terms of cryo-EM impacting healthcare? We talked a little bit before about how access to this technology... Are hospitals going to start to have to have cryo-EM microscopes? Or what's that look like?

Cameron: I don't know if they'll have microscopes much like they send out lab work, but I certainly think, if we're discussing precision medicine, and thinking about personalizing that to an individual patient, what the care plan looks like, I think certainly having a 3-D view of what's going wrong is going to become valuable, and I think it's going to become necessary. As we learn that it's insufficient information, people are not going to want that gamble. Insurance companies are not going to want that gamble.

Deb: Yeah, a three-dimensional plan. Everything gets so much more informative when you take it into 3-D, like 3-D mammograms, for instance, or 3-D ultrasounds, having 3-D analysis of molecules.

Cameron: Yeah, so it's not your 2-D readout of your genome. You're seeing the three-dimensional product of your genome. And of course, you're going to use that 2-D readout to implicate, "Oh, where do we spend our time looking?"

Cole: Right, sort of step one.

Cameron: Step one, but now, what does that look like? How aggressive does this cancer appear that it's going to be? And you'll get a lot of that information.

Deb: I think there's also lots of opportunities for biosensors implanted in areas where you know we're susceptible to cancer, and they're coming back. And Cameron works in a very interesting area, where these cancer cells, zone in on that little segment so you could maybe catch on a sensor in your phone to understand if things are coming back with your exosome work, right?

Cameron: Yeah, yeah, exactly. So exosomes are essentially little terraformers.

Cole: Terraformers.

Cameron: Right, so it's technically tissue-forming, but they're going out and developing a pre-metastatic niche that's appropriate for cancer cells. So you have these little vesicles that are released by cancer cells that carry anything that you can imagine within the cell. And it's also used by cancer cells to expunge chemotherapeutics from cells. Anyway, so these exosomes, which carry anything, so you can think of them as little FedEx trucks, or little tankers, that then go and travel and create niches that are appropriate for cancer cells. So then the metastatic march begins, and cancer cells migrate down, and then that is when metastasis happens. So I think the future with liquid-cell and cryo-EM both is really capturing those exosomes as they're beginning their travel, because then you can start looking at treatment plans before your cancer shows up to a new site.

Deb: Yeah, if you could capture that on a sensor in the body, you'd have information faster toward treatment.

Cameron: And you'd know where.

Deb: And where.

Cole: So you're talking about once... This is for somebody who has already been diagnosed with cancer-

Cameron: Correct.

Deb: [crosstalk], and perhaps [crosstalk].

Cole: And having something implanted in their body that talks to the phone in some way, for-

Deb: Yeah, people are already detecting blood glucose levels for insulin delivery with cellphones. This is just taking it to another level with sensors for cancer that could be controlled simply with your cell phone.

Cole: Wow. And-

Deb: These are futuristic ideas. These aren't-

Cole: Yeah, got you.

Cameron: Right, very futuristic, yeah.

Cole: Well, does the 3-D image apply to the exosomes, or no?

Cameron: Yeah, absolutely. So before we know what we need to sensor, so we know exosomes are important in the development of cancer. But right now, we pay attention to their inside contents, so that would be like if we were just talking about airports. So if you were at an airport, and instead of having people go through scanners to detect if they have harmful things on them, if you sat them down and were like, "I want to know what's inside your head." And we're going to have everyone go through a quick mental health appointment to decide whether or not you can proceed. That's totally ineffective, right? So we're not able to effectively just look at the insides to then catch this disease, because there's lots of exosomes that are healthy in your system, so you need to be able to sort through the good and the bad. So I think using cryo-EM to establish that pattern, so that we can see on a more global level, on a pattern-recognition level, on a visual level, what these exosomes look like that are metastatic.

Cole: Right, the bad ones.

Cameron: The bad ones. Then on that global, bigger scale, then you're able to then apply the things that you know, such as "Exosomes are involved in metastasis," to early detection and understanding where the disease is progressing.

Deb: Yeah, disrupting the cancer supply chain.

Cameron: Yeah.

Deb: [inaudible].

Cole: Got it. Jen, what do you see coming down the pike in the field of cryo-EM? And maybe from the materials side as well as the biological side.

Jennifer: Well, what we've been trying to do is, we have a new cryomicroscope called the CryoS, that we added some materials science functionality onto, like spectroscopy. So what we're looking to do, is look at some of these biological samples and do some elemental mapping to... Let's say you have calcium or iron or some biologically relevant element, and we may be able to map out where those clusters of atoms are that would give us some additional information, rather than again just imaging, but actually figuring out what type of atom is sitting where.

Deb: Or in DNA, for instance.

Jennifer: Yeah.

Deb: Inside of a protein.

Cameron: Oh, man, I can see that being hugely important to-

Deb: Yeah, its atoms are different [crosstalk], yeah.

Cameron: All things like dental health and repairing things.

Jennifer: Yeah.

Cameron: Yeah.

Cole: So dentistry, you're saying?

Cameron: Well, just like dental, like-

Deb: Calcium phosphate.

Cameron: Yeah, like calcium. So we've talked a lot about cancer, right? But there's a few unique cells that we have, and one of those is bones, which are able to regenerate themselves, and how that happens and how it solidifies and then breaks down is something that we don't really understand, so I could see what Jen's talking about playing a huge role there.

Jennifer: Right, so I have this technique that I can apply, and if I tell you about the technique, you'll have some ideas as to where to best utilize it at.

Cole: Very cool, yeah. It just continues, right? So what are next steps with you sitting at the table right now, like next steps with what you're working with right now today?

Deb: Break the resolution barrier in liquid-phase electron microscopy, and then get atmosphere system in place, so we can look at, for instance, how a virus would transmit in the air, live with a gas chamber inside the electron microscope, things along those lines.

Jennifer: Yeah, and we're thinking about what's the next microscope that would be good for Penn State, so we're looking at different options for different new cutting-edge microscopy methods that are out there, that we-

Deb: The latest and greatest technology.

Jennifer: That we may want to apply, so.

Cole: And so what, once a year or something, all the microscope people come together and you get to share your best practices and learn from others, right?

Jennifer: Yeah.

Cole: So it's a community that's international, I imagine. Is the United States leading? We must be leading on this. We talked about this building being the best building and all that, so we get to share-

Deb: Microscopy's a very international, collaborative type of science. There are lots of wonderful groups in Germany, wonderful groups in Japan, wonderful groups in U.S. or U.K. There are a lot of leading sites, but U.S. is definitely among them.

Cole: Right on. I'm going to close with a question I always ask my guests, and that is: what advice would you give to others who are just trying to break out of their one little narrow field? That's what the Huck's all about. That's what you folks are about, collaborating back and forth with one another in convergence and with everything you do, sharing your knowledge across disciplines. What would you tell others who want to break out of their little box and collaborate across disciplines on something?

Jennifer: One of my big things is just trying to learn the language. Being a materials scientist, I need to learn some basic biology terms, like "What is an exosome? What is a lipid?" These are things I don't know, or the length scales that they talk about that is different from the way I talk about length scales or concentrations, things like that. So for me, language is a big deal.

Deb: Dream big and be bold. Don't let people tell you, "Oh, yes, but the problem is, as you see." When anyone starts to comment about your research with that phrase, just go talk to someone else.

Cole: God, yeah.

Deb: Yeah, just be bold and persistent.

Cole: Dream big, people. Be persistent.

Deb: Yeah, absolutely.

Cole: Sounds like good advice. Cameron, what would you tell folks?

Cameron: So I'm trying to think of how to phrase this, but I think it's, pursue what you're doing passionately, but be open-minded to how that can be applied. So for example, one of the biggest breakthroughs that was made in Alzheimer's was NSF-funded research, so very basic science research, on how songbirds regenerate their songs each year. And so I think about that, and that was just somebody who was really interested in birds and how they come back and learn their musical talent and calls. And then it turns out that that applies to other neuro problems that we're looking at in a different way, so I think passionately pursuing, that person wasn't seeking out to be translational, but being open to that idea. So if this is your interest, you'll be fully captivated. You'll be able to do those tedious things and focus with the need you need to focus. So I haven't quite gotten there yet, but working on it.

Cole: Got it. I love that, so do what you're passionate about, but stay open, open to sharing the knowledge, open to other people doing other things with [crosstalk] bird songs.

Cameron: Yeah!

Cole: Fantastic, I love that! Well, that's a great note to close on. I want to thank you all again for being on The Symbiotic Podcast with us today, and I wish you the best of luck with all your endeavors moving forward. Thank you again.

Deb: Thanks so much. Pleasure to be here.

Jennifer: Thank you, yep.

Cameron: Thanks, Cole!

Cole: Cheers.