Oh, there you go. Nice to see you. Hi, how are you? How are you? I'm good. Well, there. Nice to see you. If you want to gather around, we'll have a sandwich here for you. Thank you. Get over here. Pretty one. Pretty one. Thank you. You're welcome. There was a little girl back in Eureka. Well, it's not going to happen. It's not going to happen. I just received an annual report from the college. Well, I... Mr. President, Mrs. Penn has some photographs that you think you'd be interested in seeing. From the Zygars, if you will report the Zygars first. This is a real old one, right there. My mother wrote these articles. And she was writing her newspaper there. Yeah, that's my brother right there. I'll be there. And I can't find... We can't find any more, but I know mother has a number of other ones. And we're going to look through her things. And if you'd like to have more, you can find them. We'll get them to you. All right. Got it. Thank you. And I'd like for you and Mrs. Reagan to have this. I'm an artist. And this is... Yeah. My brother and I painted together at Cannon Beach in Oregon. And so I'd like for you to have this. It's a haystack rock. Which is the famous one at Cannon Beach. Well, thank you very much. We're very happy to have you. Thank you. Thank you. This is just... Oh, thank you. Oh, I'm wonderful. Thank you so much. Thank you, Mrs. President. Thank you very much. I can't read my clippings. That was for when you were going to be writing. That's what that book is. Excuse me. Yes, sir? No. No, there's the press. Well, let's see. What's this? Can you let me know why? You're bound. Chicago, your food and your people. Well, I'm born there. You'll never get over that. First American born president that we've ever met. I don't think it was part of it. Granted, it was part of it the other version. Here's that set that we haven't taken. It shows it's the best description. Well, thank you. I can't match that. By the way... That's a little more modern pen, and that's a souvenir. Thank you very much. Thank you, sir. Thank you very much. Can I go after this? Wait this way. This way. I'll take care of this. Thank you, sir. How do you miss my husband, president? Hello. That's what he is. You go first. What was the president? Hello there. Mr. President? Yes. Mr. President, this is my wife. Mr. President, hello. My daughter, Mickey. Nice to meet you. Hello there. That's my son. Hello there. Nice to meet you. Mr. President. Mr. President? Hello. Nice to meet you. Mr. President? Mr. President? Nice to meet you. Mr. President? Mr. President? Mr. President. Mr. President? How are you? I'm fine. I'll get on baby. Well, Mr. President, I'll just introduce this very briefly. As you know, the inspector general's presented their fifth report to you the other day, and delivered $22.3 billion again in cost savings and avoidances. And last year, we lost one of our top inspector generals, And Mr. Bucher is here with us. It was a sad loss for us. But what the Inspector General did was they did get together and decided that in the honor of Paul that they would give an award for the top performance for a year to somebody within the IG community who had delivered most on your promise basically to reduce waste fraud and abuse. Larry Cressy is the award winner this year who is the Associate Inspector General over at the Department of Transportation. And what he did was he found that there was bid rigging in an airport construction project. It was an airport with him. And he thought that this might be going on throughout the federal government. And yesterday in the Washington Post if you notice there was a machine, a computer that was developed to show for automation, analysis, and auditing of bid rigging. And it was a result of much of the work that Larry did. And construction projects went down 10% in cost as a result of his effort. He came up with 188 indictments, 134 convictions. And so we're very proud of him. And he is our first award winner for this year. And an honorary runner-up is Bob Martin who is in the back over there. And Bob is with the Department of Pud. And as an auditor over there he's been working on the internal controls. And he's been doing super work over there. So we're very proud of him. And beyond this day we're here to honor Larry Cressy and Bob give the award. Well, I'm going to use that here because I just have some notes I'd put down here. Make sure that I... You know, there's always... If you don't have something like that, I found out there's always three speeches that you make. The one you intended to make, the one you make, and the one on the way home you wish you'd make. This avoids that and gets it down to one. But I am pleased to award the Paul Boucher Award today because it honors a basic principle in which I believe very strongly the application of individual initiative and achievement for the betterment of the community. And restoring the American people's faith and respect in their government was one of my promises. And together with public servants like Larry Cressy working hard in the inspectors general offices of the departments and agencies we're keeping that promise. And Larry, you've demonstrated accomplishment and dedication resulting in actions of national impact. And I know, though, that this award has an additional special meaning, however. It honors Paul Boucher, known for his outstanding contributions in IG and law enforcement communities and his tragic death a year ago this month, saddened not only his loved ones, but also those who knew of his extraordinary work in the battle against fraud and waste. And I know that this award has a meaning to you personally since you and Paul were friends and colleagues for many years. This award conveys the administration's total commitment to achieving our goal of an honest, effective government. But it also encourages other government employees to aspire to the high standards of service that Paul Boucher represented and which you have met. And I'm also happy to present a letter of recognition to Robert Martin for outstanding investigative efforts and expertise which is making inroads against fraud, waste, and abuse in the federal government. So I want to congratulate you both. Wish you well. Larry, congratulations. Thank you very much. Thanks for all that you've done. Thank you very much. So pleased to be able to do this and knowing what it represents and what it means, what you've all been doing in this subject. That's why I remain more optimistic than some of the people that don't know all that's going on. I know things are getting better. They are, Mr. President. The easy-high is looking. There was a great concert when you started. I just received an award myself, not an award, really. No presentations, gentlemen, who makes the quill pens for the lawyers who appear with a set. And I didn't know whether to tell him or not. I have a Hollywood story about it. Do you remember back when the great English actor was making all those pictures at Warner Brothers, where I eventually ended up in a contract where he played Disraeli and played all these characters and prime ministers over the past? Well, every year in those days, the studio used to send salesmen out to kind of tell them about the new product and find out what they wanted and what kind of pictures they wanted. So one salesman went to a chain of Southern theaters and came back with this word that when he'd asked them, well, you know how the picture's done and what would you like to see? And the fellow that owned the theater said, well, there's one thing I do know. I don't want no more of them pictures where the hero writes with a feather. Well, thank you. Oh, you look terrific. Well, thank you very much. This is so great of you to take time on your busy day like this to see me. Well, happy to do it. Oh, this is great. Hello there. Just a good to see you. It's been two years. It's your turn to come to our house. You've got to come to our house next. Okay. Can we work that out? Come over here. This is so great of you to do this for me. Oh, pleased to do it. All the family says hi. My mom says hi. Phillip says hi. Oh, he's doing real good. He loves it. And Cheryl is her hub. She's from the U.S.S. Coral Sea. And so, just moved to Chesapeake, Virginia. And our son, Paul Jr., is doing well with his kids. Linda out in Las Vegas. Scattered all over. How's the first lady? Just fine. Did she have a good birthday? Yes. That's good. We were at the ranch in California. We observed the birthday about four days early. Oh, you look so good. What? That's because I've been to the ranch for a few days. Oh, you look great. You're such a doll to do this, really. No. We've got five grandkids now. Did you believe that? Five grandchildren now. Well, for heaven's sakes. Isn't that something? Just like 41 years. Very good. No. I can't tell you how great you are to do this for me. I just love you.  There must be some reason why you've been so special for all these years. Oh, I don't know. The president's been very loyal to me. I know that. Well, it's been a pan-a-nink relationship. Sure is. Boy, what a pan-a-nink relationship we've had. Spain, all this. Did you realize that nine years would be 50 years? I'm afraid to count. Sounds great, though, doesn't it? I'd like to be able to celebrate 50 years. Oh, yeah.


From London, England, it's theCUBE, covering Coupa Inspire 19, EMEA. Brought to you by Coupa. Hey, welcome to theCUBE. Lisa Martin on the ground in London at Coupa Inspire. Because I'm in the UK, I have to say, you know of Sting, right? Well, guess who's here? Somebody cool enough to go by one name. It's Chandar, the CMO at Coupa. Welcome back. Great, Lisa, it's great to be here. So this morning kicked off with Rob's talk all about community. One of the interesting things about Coupa is this community that you guys have built. Talk to me about, I know $1.3 trillion of spend is going through the Coupa platform, the community. Talk to me about how you've cultivated this community. Yeah, it's a great question.  People buy tribal feelings. And if you look at like, you know, if you look at a product like Harley Davidson, you know, anybody can go buy any bike, but people are not buying the features. They're buying the tribal feeling of being part of that community. If you look at a product like Peloton, you know, people can go buy, who have any stationary bike or any workout bike today, but they want to be part of that community. And as my wife tells me, Sephora, right? I don't have a lot of experience with that. She's right. She is, okay, great. Thanks for the endorsement there. But again, it is about being part of the community and people like that and stuff. And that's what we're doing is, it's not the features or the capability, it's the community, the tribal feeling. And that's what Rob was talking about, the inspirational attributes of these different people that are part of this community and how we are building this community by showcasing the great leaders and their attributes and how they're transforming change in their organizations. And that's what we're creating in this conference, the feeling, the hashtag emotion of I want to be part of this cool club. And that's what we're doing. We know a lot of companies talk about customer first, customer centricity. The community here is really helping Kupa innovate on its own technology. Talk to me about some of the things that, since we last spoke a few months ago, that have been inspired by the voice of the community. Yeah, so there is this concept of co-creation that Rob talked about today with our community and a lot of the community is inspired by the community and it's for the community. And we have a number of innovations, AD plus innovations that have been inspired in the last one year by the community. And even a concept like Source Together that Rob talked about and the idea of Source Together is how can we come together as one community and drive the best negotiated savings together with a supplier. This is an idea that's been co-created with the community. So there's a number of different things. You look at community intelligence, Rob talked about commodity insights, as well as a number of other capabilities that we are showcasing today has been driven, co-created, inspired by the community. And that's what great, you want to set the innovation agenda for the industry by having this community inspire us. In fact, we had our customer advisory board in every conference that is happening to us and that's what drives us to a lot of the innovation that we do today. Speaking of customers, Rob shared a lot of examples during his keynote this morning. I lost count of how many of your Coupa spend setters were mentioned, other customers all with very strong business, measurable business outcomes. I know tomorrow in your keynote, you're going to be talking with a number of customers. But some of the things that are interesting about what Rob shared is these are examples that aren't just about refining procurement and reducing spend and it's much more transformative. Give me some of your favorite examples of where this is beyond improving procurement. Yeah, it's a great question. It's a great question. And we have a number of stories. For example, tomorrow in my keynote, I'm going to be talking about storytelling. I'm going to be talking about how we can inspire the storytelling, how can we inspire the community with storytelling. And great storytelling starts with great storytellers. And these spend setters, and we can see them in the hallways here, we've profiled about 15 of them. And they're all great storytellers for one reason. They have great transformative stories in businessmen management. But what makes them a great storytellers is they're telling a story beyond the boundaries of businessmen management. Let me get a couple of examples, right? So one story that I'll highlight tomorrow is about Yarko, the CPU of Talia. Now, I don't know if we know Talia, but 60% of the world's internet traffic goes through Talia. 60%. 60%. So if you're every day morning, checkinoutcooper.com that we all do. Every day. Or looking at some less popular sites like Facebook or LinkedIn or anything else. You're probably on the Talia network, especially in this part of the world, right? And their challenge, their businessmen management challenges, they're pretty fragmented across the Nordics and the Baltics and other regions. And now with Yarko, he's a strategic crusader, not a hired gun, but more of a driven crusader was coming, transformed the sourcing function, made it more strategic, consolidated seven systems into one system with Cooper, and had 20,000 employees using that, as well as all the different people for sourcing so that they get the global benefits of scale across the regions. Now, that's a great businessmen management story. But what makes him a great storyteller, he's telling a story beyond the boundaries of businessmen management, because he's not talking about savings attainability, he's talking about environmental sustainability. And the story he talks about is what their initiative at a board level is, zero emission and zero waste by 2030, and how the work that his team is doing is directly impacting that board level initiative on how they're driving a communication strategy across the supplier base to get their environmental plans into the Talia's operations, and how he measures plans and progress of every supplier in their CO2 emission, and how that's going to be an explicit pause on how they work with the suppliers, and how he's a trusted advisor that he is actually challenging everybody to rethink this whole idea of source to pay. So that is telling a boundary beyond the boundaries of businessmen management, it's telling a story. So that's one example, right? Is that a gentleman who's in procurement and finance within that organization? He's a CPO. That is having an impact on the sustainability footprint of the company. That's right. So directly associating with the initiative at a board level, right? So he shifted it, by shifting the storytelling from talking about savings attainability to environmental sustainability, he shifted the perception of the organization from something that's operational to something that's very strategic in the organization. So that's one good storytelling. The other one I'll highlight as an example is Mateo at Global Fund. Now, the Global Fund is the world's largest financer of fighting malaria, AIDS, HIV in 100 plus countries. They disperse $4 billion every year for that. And they have this product called Wombo.org, it's powered by Koopa, that Mateo and his team are doing. So he could tell a great businessman management story. You can say, you know, I've driven distilled transformation, I've done 99% of my POs are electronic, and I've come to this new age of where, you know, on-contract spend is being done, et cetera. Now, what makes him a great storyteller? He's telling that story beyond the boundaries of BSM again. He's talking about a story of how this, the work that his team is doing is directly impacting saving 32 million lives. How they're treating millions of people to get the right treatment for HIV. How pregnant mothers go on HIV, to get the right treatment on time so that the babies don't get infected with HIV, and how they're distributing hundreds of millions of mosquitoes throughout the world for preventing malaria through this Wombo tool that's powered by Koopa to get the right medication on time. So that's millions and millions of lives, but the speed and ease of every single medication to get there is an impact on the life of that person. And that's the story he's telling. This is so interesting because it's so common for businesses to tell the common success story. And a lot of what Koopa shares with customers holding those big white cards with big numbers of what they're saving are very impactful. What was the idea behind the Spend Center's program? Because when I was reading a few of them in preparation to come here, it seemed like it was a little bit more about the person and how that individual has facilitated transformation. Tell me about the concept. It's a great point. If there's two components to it, right? One is empirical, two is emotive. And if you look at both concepts, one is the empirical value that, yes, ultimately Koopa is about driving value. And that has to be as a company, as a capability of driving value to our customers. And that's the empirical value of you have driven so much savings, so much percentage of Spend, and millions of dollars, billions of dollars savings, et cetera. Procter and Gamble, for example, 2.5 billion dollars in savings. That's the empirical value. It's very clear that's the value. But behind that is a person. And that is the emotive story of what is that person? What is the personal story? What have they gone through in their life? What's their nurture and nature? And how that's influenced them, that's made them into the great leader today. And that's the emotive stories we're trying to also tell on the Spendsetter side. So there's the value side of the story, and then there is the emotive side of the story. And the Spendsetter.com is purely on telling the human stories, because behind every purchase order is a person, and we're telling the story of that person. So as we look at the changing role of the chief procurement officer, the changing role of finance decision makers, not just here in the UK, and I know Koopa recently did a study that showed that 96% of UK financial decision makers said, hey, I don't have complete visibility over all my spend, so big opportunity there. But even from a transformation perspective, the Spendsetter's examples, how is that showing that Koopa can fundamentally help a business, not just change procurement, but have such wider, wide lasting impacts? Yeah, I think ultimately, if you look at procurement, for it to go as it's going from operation to strategic, it is getting that seat at the table. And getting that seat at the table in any executive discussion is about first aligning to some strategic initiative that is important at that executive table. So more as we align these value stories and the value that procurement is driving to these strategic initiatives that are important at the board level, at the executive level, the more the profile and the more the RESPCT. So you like to say, and get that seat at the table, and that's what this whole Spendsetter's program is aiming to do is, A, showcase the personal heroes, and B, showcase how they're telling stories that align to bigger level initiatives that's getting them to elevate their position and get that seat at the table. And that's what the plan is there. So lots of growth, second quarter results, I was taking a look at those revenues up, billings are up, very high renewal rates. So from a customer satisfaction perspective, the data is there to show that Coupa is going in the right direction. From your perspective, how influential are your existing, your incumbent customers in helping prospective customers evaluate Coupa and go, this is the right decision for us? It's a great question. I say we live in a peer-bound world, where it's really, more and more, first of all, 80 to 90% of buyers' journeys are self-directed because buyers are more powerful than ever before. And second of all, in anything we do within our consumer, in the personal lives, as well as business decisions, we rely more on peers and people we trust to help us make those decisions. From that perspective, our best sellers, the best sellers we have in this conference are our customers. I just came from an executive luncheon where we had 50% of the room was customers and 50% of the room was prospects. And we had our best sellers, not our salespeople, our customers talking to the prospects in real, authentic conversations of what's value, what's their journey, what did they struggle with, and what are the lessons learned, and how did they get there? And those are really meaningful interactions that ultimately is going to make a prospect, influence a prospect on what decision they have to make. Right? So that's very, very important from us and providing a platform for this authentic dialogue and this authentic interactions, right? That's important for us. And also I think ultimately in a SaaS business, the true measurement of success, I say is two things, right? One is what I call lifetime value, and two is the number of brand advocates. So the idea that if someone is staying with you longer and giving you lifetime value and is shouting from the rooftop that I really love my interaction with this brand, then invariably you're driving value to them in a long-term way. And that's really the true measure of success and that's what excites us from our perspective. And is the foundation of that trust? The foundation of that is two things. It's trust based on value, right? And you've got to deliver value. And Ram has a great line where he talks about it is not about customer satisfaction, it's about customer success. Because many times the customer may be satisfied, may not really know what their success metrics really mean, but it's not about sometimes the customer may not be satisfied, but really be successful because you're driving the true metrics what is important to the customer. So once you get the value delivered and do it in an open, authentic way, then in that case, there's trust that build and based on that trust, you earn that trust and that becomes the foundation of the lifetime value. We were talking about, well, Rachel Botsman was talking about the importance of a brand, any brand earning trust. I loved how she gave that example in her keynote where she showed three brand logos, Uber, Facebook, and Amazon and said, trust is so contextual and so subjective, but clap for which brand you trust the most. And it's so interesting when she started talking about Facebook got the least. In fact, Facebook got no applause at all. I was expecting a few folks to maybe do some clapping, but Amazon being the clear winner and I thought, yeah, I trust Amazon to deliver whatever it is that I buy when they say they're going to deliver it. And she said she trusts them to do the same, but would you trust them to pay their taxes on time? So when she started talking about trust being subjective and contextual, it really kind of changes the whole dynamic. So that's earned trust, but also the ability to reduce the risk that your customers are facing, whether it's overpaying suppliers or paying duplicate invoices, that trust risk balance seems pretty critical as well. It does, it does. It's an interesting perspective. I think because in the case of Amazon, I think there's operational trust that they're going to get the job done and deliver the whatever you ordered in one day with Prime or two days with Prime. So there's operational trust, but is there a trust in the sense of purposes where she was going with, right? And today for organizations, especially with the Millennial Crowd as being customers as well as employees, the question is you can get operational trust, but do you also have a sense of purpose that they trust in and have that and be authentic as an organization? And that's where I say it is not being, we talk about AI as artificial intelligence, the real AI is authentic interactions. Authentic interactions. And that's really the authenticity as a brand, being open and acknowledge your failures, but strive for excellence for success and have this open platform with your customers and always look towards adding value. I think that invariably over time creates this trust feeling that ultimately drives long-term lifetime value for us. So that I think is the most important thing.  It's great. So I have three, one Procter & Gamble, a company that my mom knows about, my 86 year old mom, so one of the greatest brands. So that's a great story about again, they have a great business management story, but they're telling a story beyond the boundaries of business management and that's a fun story. And then we're going to have the global fund. Again, I told you, one of the world's largest financial fighting HIV, malaria and AIDS. And we're going to have Telia, one of the largest telecommunications providers. Excellent, so really kind of showing the breadth of the technologies and the industries that Coupa helps to transform. And the breadth of the personalities and the people behind that are driving all this change. Excellent, well Chandar, thank you for joining me on theCUBE. I wish we were going to be here tomorrow to see your keynote, but it sounds exciting. And the Spence Center program is certainly one that I think is quite differentiated in terms of telling those transformative stories that you said are both empirical and emotional. Yes, thank you Lisa, it's great to be here. Likewise, for Chandar, I'm Lisa Martin. You're watching theCUBE from Coupa Inspire London. Thanks for watching.


admin can merge student accounts together from the Students tab in the admin dashboard. This is helpful when merging student accounts that either both have student IDs, both have email addresses, or if the student is not showing up in the Assign Missing Student IDs page. To merge two student accounts, first sign in as an admin at app.csaw.me on a computer and your preferred browser. Next you'll click on the Students tab. Then you'll search for the student you would like to merge by name using the search bar or by scrolling through your student list. When you have found a student to merge, click the Edit Student button. Then click Student ID. If there is already an ID entered, delete that ID and replace it with the correct student ID. Press Enter on your keyboard to merge student accounts. When merging accounts, please double check the display name to ensure that you are merging two versions of the same student. Merges cannot be undone, so it is important that you do not merge two different students together. If you are merging two accounts that both have email addresses, select the correct email address that you would like your student to sign in with. Click the green checkmark and follow the prompts to complete the merge. After the merge is complete, your student will have one cohesive account. Repeat as needed. Thank you for using CSA.


Many of our people in the far north tell the same legend to our people but it sounds so much better in Dene than when it's translating it, but this is the way the legend goes It was winter and many native people Were camped together one evening to hurt a little baby crying by the lake shore Some young girls ran to look for the baby But as they came closer to the spot the crying stopped this happened three times The fourth time An old lady came to see what was the matter She walked around trying to find out where the song was coming from It seemed to come from in front of her But she could see nothing but dear droppings When she's gathered them She saw a baby which was only about the inch a few inches Like he said You know your thumb glove that was the size of this baby She picked him up and put her him in her mitt and Went back to camp the old lady Had several sons But she decided to raise this little boy as well and call him that's an A&S shot One day her sons went hunting When they returned But Sunayana shot asked his grandmother for the front feet of the smallest deer That his uncles had killed The boy was given the feet to eat and he was so happy After Several another successful hunt the boy once again asked for the front teeth of the smallest deer The hunters would not give him to him for he They wanted the rest of their feet for their own children And he was offered another part When the grandmother returned with Without the feet, but Sunayana shot was very angry And you see how angry he is here Yeah What did they tell you but Sunayana shot asked his grandma His grandmother replied they said you were not the only child in the camp who wanted them This time you have to have another part of that deer But Sunayana shot just because says just because I'm small They have insulted me Now I will starve them when the people heard this they became very angry We will see if the boy can make a start They said the next day They moved away leaving but Sunayana shot and her the grandma all by themselves But Sunayana shot told his grandmother to cut some small branches and put them in the fireplace for abundance of each of the abandoned lodge When they have completed the hunt She was to tell him a little later The grandmother came to him and said the branches and your uncle's lodges Have burned in the shape of deer hooves And all the other lodges the remains are round little stones This means that my uncle shall always have deer, but the others will only find stones And they will soon die The grandmother was so also afraid of starving But Sunayana shot saw This and he says to his grandmother as long as I can be with you. We will never starve She lifted them out on her back and they set off the search of food By the middle of the day They came to a small muddy lake Stop here and fish But Sunayana shot told his grandma And his grandma said here in this mud Why there's nothing but worms in this swamp His grandmother said she lowered the fish hook into the water and a large Jackfish came up But he didn't bite but Sunayana But Sunayana shot said lure the hook again grandma She did and so caught another Black trout That's good. He said now. Let's build a lodge. We will camp here and prepare the fire for make some dry fish She did The boy went The grandmother began to cry and she was all alone Then she heard a little noise out there from the lodge NK, but Sunayana shot all covered with ice Grandma, but Sunayana shot says to his grandma. I was afraid that you left me, too Grandma as long as I'm here. We will never Starve and I will never leave you. He told her to take off his belt Inside His coat she found many deer tongue tips He had killed the deer by biting off their tongue What looked like ice covering him was foam from the mouth Remember that Sunayana shot was just very very little and that was the only way he could kill The next morning he says she says he says to his grandma Let's wait. Let's go where I had killed the deer The first one we see you will dry and pound for me Gather their grease, but don't eat any yourself yet grandma They set off and soon came to a lake there on the ice lay the deer The grandmother got busy setting up the lodge preparing for to prepare the meat The boy went away to look for his uncles He found their lodges on the other side of the lake The two uncles sad fishing there was no other people there Only the families that his uncles had left But Sunayana shot hit himself Took off his new shoes Turned into a deer Then he walked out there where the hooks lay in the water He pawed the ice Softly and then the uncles Saw this and said that's strange That little deer is playing with our hooks says at one of the uncles Do you remember that queer little boy our mother had? Maybe it's a medicine man and he turned himself into a deer and now he's here to laugh at us Let's track him When they looked up and saw the small deer had disappeared They followed his trail into a patch of pine trees There the tracks stop and the snow shoe marks on the snow Following they came across the lake where this saw the spruce lodge Then they found their mother that's an a nation and that's an a nation They also saw the piles of meat and fat Soon after the arrival of the Sun's the old Lady the mother felt sick and she passed away That's an a nation turn into a deer again and disappeared So you see no matter how small you are That you think you are You can do just about anything in life So there's a lesson in everything that the legends tell us Legends teach us a lesson Stories have an ending When you tell about Cinderella there's always an ending ah Went off with the prince and left happily ever after but legends They tell you a lesson of the things and life itself So my teacher for listening to me


OK, zelo smo poču. Vsega lektura na tvoj morač. Mi je prav, da se prof. Dan Kožok, z nesetnjih rečenih v Italy, prišliš, da se prišliš o optikljovje, basične in aplikacije. Vse, poč, prof. OK, možete me zelo? OK. Dobranje, sreče. Vsreč sem izgleda površenje, tako da se površenje površenje. Vsreč smo tukaj, Trieste, downtown. Eletra synkrotrona in vse elektrolazir. Vsreč sem izgleda, in vse vse vse vse vse vse vse vse vse vse vse vse vse. V Trieste vse iz ICTP vse je universitet, vse je tukaj scientific pole, in vse je elektrolazir in vse je vse vse elektrolazir in vse je vse vse vse vse vse vse vse vse vse vse vse vse vse vse vse. Vse osvej je also v ekraniji temperat optizovi blureče v Boballot. za delsaj Ok. Vsreč sem seating. kako je je tukaj od odveči, kako je zespešnila obtikl kako je verišnja izdesna v obtikl tukaj, nekaj posled够im po vzivnih medzije o obtikl tukajs, o reč眼 na rečie, čez pravda na našje odvijsce od obtikl tukajs jutroga in moždelje, moželje, smešljenje, vršenje, vzdej, in vzdej, govekih, in vzdej, in vzdej, in vzdej, aki večnih cilu, i papierin, i vents, i tanko, i začonje vzdej, zelo, da je zelo, in je zelo, in je zelo. Zato smo počučati z radiečnimi priježenimi však. Zelo smo však vzeli, da je potom energija, in je momentum. Vzeli momentum je tudi transferit do objev, z vsem, da je zelo, z vsem, z refleksijem, reflekšnje, as you will see in a moment, then if we consider a light wave being carrying a momentum flux, the radiation pressure is the momentum transferred per second per unit area, or in other words energy deposited per second per unit area divided by velocity of light. So the point is that this radiation pressure exists, what is the effect? The effect in terms of forces is difficult in general to be detected because these forces are very small. But with the use of laser light, which is able to generate high optical intensities and high optical intensity gradients, we can observe the effect of these forces. So just to point out that Kepler probably was the first to observe the radiation pressure effect on the comets, on the tail of the comets, observing that they are oriented, they always point away from the sun. So, saying that the particles in the tail of the comet are oriented because of the sun radiation pressure. The radiation pressure of the sunlight on the earth is, in average, at the level of micropascal, so it is very small. Fortunately, and unfortunately, we can think in both terms. Another example, more recent example, is a Doppler cooling damping of atomic motion in what is called optical molasses. So, if you use two laser beams, counter propagating these laser beams are to slightly below the resonance, you will have the atoms in between, which absorb more photons if they move towards the light source due to the Doppler effect. And then they absorb photons and they emit photons, but the photons are emitted randomly. And this means that the momentum, which is transferred by emission, it is less in the direction of the movement of the atom, and you will have a negative force, which slows down the movement of the atom. So, if you combine three, in three directions, this, you create an optical molasses, where you create a high density of atoms, which are slowed down. So, first observation of this phenomena was done by Hansch and Schullow in 1975. The year is missing, so is 1975. Now, let us discuss ray optics approximation of the effect of the ray of light on particles, and let us see how big is the force exerted by a ray of light on a micro bead. And we consider that the size of the bead is two microns, that is, is bigger than the wavelength. We consider that the bead is reflecting perfectly, so the reflection coefficient is one. And following with some simple calculations, we get that the force is about, on the bead is about 2.7 by 10 to minus 27 Newton, if we consider one photon. But if we consider 10 to 15 photons, which means a wave is a power of 0.4 milliwatt, so a low power laser beam, we get 2.7 piconeuton, which is a small force, of course, but what it is important, it is, which is the effect of this force. So, if this bead is in free space, then the acceleration, it is 34j gravitational acceleration, so which is very big, because the bead is small, the mass is small, so the acceleration is big, even for a small force. If the bead is in water, then we have a fast damping, so we have damping because of the water, and we have a terminal, so the bead is accelerated, but very soon stops. It reaches, if we stop the laser beam, I mean, if we keep it constant, we get a terminal velocity, 360 micrometers per second, which for micro world, it is big velocity, and we have the time constant, which is 0.8 microseconds, which is small. It is to be noticed in this case, that for a small size particle, damping is dominant over inertia, because the mass goes with the size to the third, while the drag coefficient goes with the size. An example from biology, I think here, not I think, I do not see the lower part of, so the second road there, but in any case, so the movement of a bacterium in water, so to have the bacterium moving in water with normal velocity of some microns per second, the motor should develop a force of about 0.51 piconeuton. So what I want to put in connection is the range of the forces that can be induced on micro particles by light, and there the range of forces specific to biology. In fact, there is a very, oh, I cannot escape this, because all the references are that row. I don't understand why. We do not see it, but it doesn't matter. So this is from a book of Howard, Howard on cell mechanics, so you will find them anyway in the PDFs that I download on the site. Just to illustrate different type of forces, and their magnitude is at the single molecule level, and we have elastic, covalent, viscous, collisional, thermal, gravity, centrifugal, electrostatic, and van der Waals. The point is that they go in quite a wide range, but anyway, anywhere here you see piconeuton, and piconeuton by the way is also the weight of a single red blood cell. So this is a good reason or if one asks why optical tweezers in biology, the answer is around, also around this thing. So now let us make a step forward, and beside reflection we consider another bead, which is perfectly transparent, so reflection is zero. We have only a reflection, so we have the bending of the ray, and by conservation of momentum we have the force transmitted to the bead. In this case, the magnitude of the force is lower than twice the coefficient. This, for reflection, is two. In this case, it is lower than two. It's clear now from this vectorial decomposition. Then it comes out that the total force on a particle interacting with an incident light beam by reflection scattering, but also refraction absorption emission is given by the difference between the momentum flux entering the object and the one leaving it. If you remember, this is a slide from Professor Markano lecture, where he was speaking about sample interacting with light and the effects induced, and about energy conservation. In this case, we consider also momentum conservation, but the picture is the same, the approach. We have incident transmitted light, we have interaction here, and if we measure the differences between what it enters and what it exits, we can get the force. This is in principle. Now we will see in which terms and when this is possible to do. Here, I want to introduce many, many references on optical tweezers, optical manipulation, but I think that kind of Bible is this book by Arthur Aschkin published in scientific publishing 2006. You can see the cover, and the cover is even very nice experiment describing. It is describing the levitation of a micro particle. In this case is a hollow micro particle to have the weight to be less, and the weight of the micro particle is compensated by the laser beam. This is the photo, and this is the scattering from the micro particle. This is a mere pattern that you can observe after the scattering from the particle forward scattering, and I think it is very nice physics here. Actually, you need only piconuton forces to balance the weight of the particle. The problem to make this experiment is here, how you detach the particle, which initially sits on a surface, and there you have van der Waals forces, so you have small forces, but much bigger than what you can develop with radiation pressure. So instead of piconuton, you have nanonuton, and then he put a shaker, a piezo shaker, to detach the particles, and then they are brought to the equilibrium. We did this experiment also in the lab. I think this is an example of an experiment that can be done with very low cost, and it is very nice, I think, as physics, because you have here acoustics, you have optics, you have a lot of stuff for students, but the book is very, very nice. The advantage of a book is this. From a paper you get information, and what one succeeded to do. From a book you get information also about the failures, and what one learned from the failures, from when the experiment didn't work, which is, in my opinion, very important. Now, again, I come back to the ray optics, just to illustrate the origin of the scattering force for a plane wave. We have only scattering force, so we have only a component that pushes the beam forward, but if we use a Gaussian beam, a laser beam is Gaussian beam, beam 0, 0, and we have the particle out of the axis, we see that there is a force called transverse gradient force, this component, which brings the particle towards the axis. If the particle is low index, for instance, bubbles, air inside a lipid shell and outside water, means low index particles, the particles are repealed out of the beam. So this is a case for a mildly focused Gaussian wave beam, which means that you focus a beam through a low numerical aperture lens. And that's it. Ashkin in 70 published this seminal paper showing what he observed. He observed that focusing the beam with low numerical aperture into a sample where you have particles in liquid, you get the particles confined along the laser beam and pushed against this wall. And this is called 2D trap. If you put another laser counter propagating, you get a 3D trap. This paper is very important because it introduces a concept, but theoretically Ashkin also hypothesizes that similar acceleration and trapping are possible with atoms and molecules using light tuned to specific transitions. Then this is 70, then it was 75, now the Doppler effect, so it is a technical stuff, then you will see. So then we skip for a moment to 86, when Ashkin and Steve Chu and the colleagues, they introduced a single beam 3D trapping, so the single gradient force optical trap. So using a high numerical aperture objective and one single beam. You can see here, so the forces and why the bead is brought to the focus of the beam. And in red I figured out also the component of the force which arises from a low numerical array. So from an array which comes close to the optical axis, the component of the force is oriented like this, which means that this will push the bead. So we will find an equilibrium. Beside this, we have also reflections, of course, not only refraction, which are not considered here. The setup was very nice, with lateral observing beside this axial, so this is the axis of the microscope, let us say, which allowed also to see the fluorescence. You see here in fluorescence, you see the scattering of the bead, the fluorescence in water. So it's beautiful paper and very important because they basically screened trapping particles from me to Rayleigh regime, so in a huge range, from 10 micron to 25 nanometer experimentally. Actually the work around this was about atom trapping. We will see in a moment. So we were speaking about small particles, small particles in the Rayleigh range. So for this, you cannot apply the ray optics, of course, you can apply the dipole approximation and you still have the same type of forces at the end, as effect, gradient and scattering. Because idea is to try to treat for all the range of particles as size, as material, the effect of radiation pressure in terms of scattering and gradient forces. So for gradient forces, we have a dependence on the size to the three for scattering from the Rayleigh scattering, we have a size to the six. It is also nice that they give the conditions for axial stability and transverse stability, from which you see that theoretically you can trap 14 nanometer particles and they trap experimentally 25, which is not bad. This is a long story about this paper. Aškin was working at Bell Labs and usually the paper had to be sent for an internal review before being sent to the journal and the internal review was very bad, was in four points. The first point was there is no new physics here. The second point was but it is not even wrong. So I could not find nothing wrong here, which is probably sarcastic saying if you do something or you do well or you do wrong, if you do not even wrong, it is bad, bad. And the third was it might be published somewhere, the fourth but not in physics review letter. So at the end this paper was sent because they discussed and it is one of the most cited 20 papers in the history of physics review letters. I might go run out of the time but I tell you also this. What we learned from this?  If you are an author and you are convinced about what you did, do not give up. So in terms of ray optics, because we spoke about Rayleigh regime, now Mier regime, where we apply ray optics, Ashkin also now developed the expression for the force scattering gradient in terms of Fresnel coefficient reflection transmission and at the end it comes out of the simple formula, now F is Q and one power divided by C, where Q is dimensionless coefficient depending on the shape, depending on the geometry. And this is in 1992. From this, he theoretically also derived which is the force exerted on a particle, when the particle is not in the center, so it is moved axially or axially or laterally. And here you have the axial, so you see that equilibrium you have zero, so Q stands for that coefficient, the Q total, so you see that if the bead moves by its radius, then the Q increases very much. And you have also some force outside because otherwise, how you explain that the particles come into the trap is not that you switch on the laser and you have the particles there, you will see in the afternoon. The same is valid for transfer sources. Now, let us go a bit back, I said your story, 70, something 75, so why the way to single beam, which was 86, was so long, not because they hadn't ideas and were not able to do, but because actually, or mainly, Askin's dream was to trap atoms and not only his dream. So there was one contribution of him, very important theoretical, on trapping of atoms by resonance radiation pressure, where he put the bases basically for what they did in 86 experimentally. So in 86 was the first experimental observation of optically trapped atoms using a single beam gradient beam and cooling the atoms with a Doppler effect. And this was the basis for two noble prizes, so one was 97, even to Claude Cohen, Tanoji and William Phillips for development of methods to cool and trap atoms with laser light. And 2001, Kornal Ketterl Viman for the achievement of Bose Einstein condensation in dilute gazes of alkali atoms and for early fundamental studies of the properties of the condensates. Well, Askin, meanwhile, focused on using optical tweezers to trap and study various living things, including tobacco, mosaic virus, various bacteria, red blood cells and without damaging them. I do not bother you anymore with Askin.  And you might notice that his technician is in lot of his papers as outdoor, which is not very common in our days. So if you want more information, you find here. So in one slide properties of optical tweezers, so what particles we can trap as material, dielectric, metallic, so I didn't spoke about metallic scattering and gradient force because there we have the polarizability that is not real, it is complex and we have also a term which is absorption and counts in trapping. And basically what is the consequence is that you cannot trap in 3D or very difficult. There are some works, one is mentioned here. Biological cells, macromolecules, intracellular structures, DNA filaments, low index, so for instance ultrasound and contrast, they are lipid shell bubbles with gas inside and they are very much used when you are doing ecography to increase the contrast in ecography. So they have a medical interest. Crystal or amorphous material, size from 20 nanometers to 20 micrometers, shapes, spherical, cylindrical, so basically I would say everything. Types of laser beams gaussian, lager gaussian. So lager gaussian, they are important besides their donut like intensity profile. They are important also because they carry angular momentum which can be transferred to the particles. Also basal beams are interesting because you can trap particles in a bottle. In basal beams, they reconfigure themselves after the obstacle. So this is why you can trap bottles of particles. Here I mentioned some reviews which I considered important and the group of others head in Denmark is known for nanoparticles trapping. Now some examples, funny I hope. So here what you will see, you see a bead and here you might see the donut intensity profile of an LG beam and this is not a bead, this is a bubble, sorry. And this is a bead stuck. So you see that the bead pushes the bubble but the bubble does not exit the ring because it is kept there by intensity and now it exits. The configuration here is this. The bubble, beer, champagne, buoyancy goes here by itself. Here we confine it and the stack bead is here and then we move to understand the experiment. Another, but here is not orbital angular momentum transfer. It's just the donut beam used. Here is a very simple rotor. So it's a Gaussian beam and a small piece of glass which is trapped like this along the optical axis and then due to the asymmetry is rotating. But then how we stop it? Using a donut beam and how we stopped because you put the ring of light on it and so on and so far. And finally this is an example of using a Gaussian and a Lagrange Gaussian because Lagrange Gaussian is produced with a diffractive optical element and in all the cases you have the zero order which is a Gaussian beam. So what you see in the center here is due to Gaussian beam. What you see here is due to lg beam which transfers orbital angular momentum to the particles and at the moment we change the direction of rotation simply making negative the diffractive optical element. We had projects, nice projects with this, mainly with bubbles because it was this problem to study a bubble not here but here. So far from surface excited by ultrasounds so we had to keep it here. So we used a donut beam and the buoyancy was balanced by the zero order. And then we excited with ultrasounds and we studied the vibration of the bubble and then two bubbles and bubble close to tissues because this has implications in medicine for drug delivery, local drug delivery. If you can identify where the bubble is and it brings also some drugs and you can make, implode it so you deliver locally. So about optical trapping I am looking, OK. Optical trapping and manipulation of bioparticles leaving cells. So the issues are this. Do we damage because there is a huge intensity of light there and if we damage which is a level of damage. This is an important question in my opinion because usually since I moved toward biology I learned this. Because before the question was do you damage it? No, I don't kill it so I don't damage it. Yeah, but if I lose one arm I do not die but I do not feel well I think. So the idea is if you damage to take care that you do not damage that function that you are going to study to be sure about that because otherwise is not good. So then the shape of particles in biology they are very different. They are not symmetric. Can we trap? I answer you first to this, to two, yes. I mean because you trap for cell, you trap through the no-close. It is enough on intracellular particle and so on. So again I said that nothing about ashkin but I'm sorry. He was the first trying to trap tobacco mosaic virus and it was a science publication in 87 and it was very nice because it was demonstrated that using even a green laser the particles were not damaged. And then the particles are very small transversally. So it's difficult to see them as image of the microscope but he used Rayleigh scattering, lateral scattering and counted how many particles when a new particle came into the trap. Nice. So what type of laser is good to use? The type of laser depends on the absorption in water because most of the material in a biological cell is water and proteins and other molecules which absorb light and they tend to absorb light toward the violet while water tends to absorb light toward infrared. So the compromise is more or less in the middle around one micron. One nice experiment that I want to mention with red blood cell is one experiment there are hundreds so I do not have time I just choose one. One keeping a red blood cell if you trap a red blood cell with one beam it goes like this. This is trapped with two beams and is investigated in the same time with Raman. Which is important because with two beams you can also stretch the cell and you can observe the correlation because the stretching and oxygenation because basically what a red blood cell does brings oxygen in the body is essential and how it delivers stretching it, modifying the shape red blood cell is very flexible. So here we spoke about two beams keeping optical tweezers, the cell. So how can we get multiple optical tweezers or by time sharing that is you move very fast the laser beam from a trap to another so fast that the particle in the trap does not understand that the laser is not there. And you can do this with galvanomirors or the acoustic deflectors. Galvanomirors are slower cheaper. Acusto are faster but are more expensive and you can get 2D arrays. With diffractive optical elements you can get 3D arrays and I think Tatjana Aliyeva spoke to you about nice optical currents in 3D that can be created with one element then you can also combine more. So from our lab what we did was a configuration like this that we looked in a capillary red blood cell from two sides. One we looked with 100 per the objective with a high numerical aperture objective to trap and we passed this through a modulator to create multiple traps and one we imaged laterally but since the objectives are big you cannot fit two high numerical apertures together so this is why one is 40 one is 100. And what you see here this is a lateral view of a red blood cell and this is the up view of the two traps and how the red blood cell moves is rotated. Can you follow? And here we have four traps and the red blood cell which sits and we want to see if we can detach it and we can tilt and also you see look here. Actually we were interested here in the following application in an application where we circulated in a capillary we circulated particles to be analyzed with X-ray so it's kind of X-ray, let us say crystallography but at room temperature for micro crystals that you cannot grow to big sizes so we tried first with starch and so we move the particles here and here we have the X-ray beam and here the detector and here you can observe the diffraction pattern for one shoot and here since the beam is focused the X-ray beam is focused sub micron level we can investigate in different points is what you see here this is a grain here here and this are the small patterns and this is a bigger pattern and then we made the proof of concept also with insulin micro crystal now measuring piconeuton forces with optical tweezers direct and indirect measurements so usually indirect methods are applied where you measure the position and you assume that there is a harmonic potential characterizing the potential of the bead in the trap but you measure the position and to measure the position you need to calibrate so you need to measure the stiffness recently it is much developed this technique direct technique with single gradient beam and this is why I decided to to speak about this first so in this case we detect the light momentum changes directly so this is basically for single beam trapping is introduced by the group from Barcelona, Farae Montes Vsategui and it starts from the idea that we should in principle measure back scattering and forward scattering to have a rigorous measurement now as you see here you see the scattering from a particle which is slightly moved from the optical trap ok but most of the light is scattered forward how much 95% so this would allow us if we use still you have to use a high numerical aperture because the angle is pi to collect which fulfills the abesign condition because otherwise you cannot skip the position you should be sure that what you measure is located where it should be and where you measure is a back focal plane and we will discuss shortly about this so the abesign condition allows you to know where let us say a ray or plane wave goes in the photo detector plane if this rule is not respected then you have variations of the positions and this is something required for aplanatic lenses or coma free lenses then the condition for the photo detector position in back focal plane because you want the intensity does not change the pattern there for who does Fourier optics it is a back focal plane it is a Fourier plane in the back focal plane you have the intensity of the Fourier transform of the light scattered so this means that if I have an intensity pattern like this it can move and the shape should remain the same which is the shift invariance of the Fourier transform then you can use a detector like a position sensing detector which is differential detector which detects the differences for the light spot moving on the detector in one direction and connect this of course with the intensity and to the force and what you get at the end is the force is proportional to the signal detected by your PSD and a constant which means that you measure directly the force through the signal not through position but is independent of shape size refractive index so it is strong is also insensitive to changes to the track shape unfortunately it requires a high numerical objective so this means that we have to put one objective to trap and one to detect with very high numerical aperture very high numerical aperture means working distance where we put the sample this is the question because if you work in biology you have I mean you need to put the cells, to put the water to put the sample so for positions close to the equilibrium there is a proportionality also with the position so in fact historically those who introduce a back focal plane not introduce back focal plane interpreted what you get in the focal plane is from the interferometry point of view from the interference point of view so jits and schmit so they gave a model and measured and said okay the interference between the light scattered by the particle and the light you have some light which is not intercepted by the particle and with this they built this model so I put first the position and they said but this is valid also for momentum then there was another step forward actually probably everyone knows professor Gustav Ante where they developed counter propagating beam optical tweezers to measure forces for single molecule experiments what was the point the point was this okay if we do not want to use we cannot use high numerical aperture lenses what we can do we use smaller aperture smaller aperture but also smaller laser diameters and then well not very small but small enough so you have this is the size of the laser beam but this is aperture that you have a disposition and this is equilibrium and this is shifted and this gives you there is a small problem here so this is a setup for measurements there is a small problem you have counter propagating beams so why such a tweezers was used and not to single beam because of the low numerical aperture so if you can make experiments only with one side it would be perfect because it allows you to work here you have space but like this you have two cover slips very close each other a channel and you insert a pipet you fix one on one bead a tether and then you have the molecule on another bead and you move the chamber and you make your measurements in fact here is illustrated one example so you do not see the DNA filament because it's too thin but you sense a force so the bead one bead is kept in the trap another one is on the pipet and you move you stretch the DNA and you see the force versus the length then there are other configurations with a single beam with as I shown with two beads one fixed and one laser beam and two laser beams I do not enter into detail is one of the good reviews is this by Mofit Bustamante and so that you can find now if we speak about the indirect method so the bead in the trap so first of all you need to measure the stiffness the bead in the trap has a potential a harmonic potential which means that the energy goes as a parabola and the force is proportional to the displacement this keeps laterally and vertically and kappa B so kappa B is Boltzmann constant and T is temperature and there is a value this is useful for me at least when I make calculations kappa B T is around 4 pico Newton per nanometer because if we use traditional units then you get 10 to minus so what you can do here you can track the particle you have the x, y, zeta bead in trap then if you make the histogram of the position distributions you get a Gaussian if you consider the Boltzmann statistics the probability density of the bead is this and then from the Gaussian you can get the potential that you get the stiffness of the trap calculating the variance for instance so fitting one of the two so it's relatively simple but you have to you have to consider that if you add Gaussian noise in your experiment this Gaussian noise overlaps on these measurements and this means that you underestimate the trap stiffness then the power spectrum analysis I will skip this because reason of time and because Suleiman will present you in the afternoon this is better so basically what you do you make the Fourier transform and calculate of the traces calculate the power and from that you get the corner frequency a parameter which allows you to determine the stiffness OK, now kind of conclusion so this is a paper quite relative paper in which this guy from Minsbruk he measures axially the force with direct method and so what we have here we have the theoretical force along the size of the bead the bead is 3 micron and here we have the units in micron sorry so it's blue then red is what is observable because anyway the aperture is smaller then if you reduce even more the aperture black is 0.8, red is 1.4 0.8 oops, sorry 0.8 you already have errors far away from the equilibrium and this black line is the linear which keeps only in this region OK so then if we think again at the force expressed as q n1 p divided by c we understand this OK so as a conclusion here typical values for optical tweezers go from 0.01 to 10 piconutron over nanometer and these values are complementary for what you have with AFM usually so it is not that one is better than the other they are complementary you can study and now we go for applications for optical tweezers in living cells and what we can do probing so if we have a bead in a trap and the cell which moves we can measure the force exerted by the cell when it moves this is one example if you have a bead and the cell which because not all the cells move so neutrophil moves very fast other cells do not move like also neurons they move but not as fast but you can probe them pushing on them indentating them and Sulejman will speak about this now this is a third example sorry you can also put some molecules on the bead attach the bead on the membrane and pull to extract teters to study additions of molecules to cell surface so is a picture about quite a lot of experiments that you can do with a relatively simple tool I give you one example from our lab working collaboration we see professor Tore they are studying neurons very young neurons two days young neurons because those neurons develop so they are because the connections are not between neurons are not established so they are neurons which develop and it is very important how they develop and how they create the synapses for all the future of the life of an individual or so so here what you see you see at the terminal part of a neuron of an axon like this which is called gross con which is like my palm fingers which are called philopodia and lamelopodia and the base of this is acting structured in different ways I show you the movement I hope you see so that is the philopodia the role of the gross con is to investigate look what signals come from other neurons that we connect or not connect and these are mechanical biochemical signals so what we did we measured forces exerted by lamelopodia and here you see the bead the lamelopodia pushing and the force exerted and here you see a philopodia protrusion it is a bundle of acting filaments and by polymerization it goes and pushes so what we learned from this was that forces exerted by philopodia were below 3 piconeuton and by lamelopodia below 20 piconeuton and I tell you anecdote about this value 3 piconeuton we were very upset about this value that we obtained because we read papers for bundles of acting synthetic bundles of acting developing much bigger forces and more than an order of magnitude and we said we are wrong with the experiment we do not measure correctly and observing better what happens we saw that the forces are not continuous so the philopodia is testing the bead and moreover is clever sometimes we say that the neuron is clever smarter than the person because if an obstacle is reached it does not push fully on it but there is a reaction which limits the maximum force that can be developed and this is sustained by by the fact that there is a high frequency of interception then we work with drugs inhibiting acting inhibiting myosin and inhibiting microtubules so that is the structure of the cell to see what happens and for instance one interesting result was that inhibiting myosin and microtubules philopodia continue to exert forces up to 3 piconeuton because there you basically have acting only there are quite a lot of papers after that about this cell membrane indentation by optical tweezers to measure cell elasticity why to measure cell elasticity or stiffness because different cells have different mechanical properties because cancer cells change their mechanical properties during their cancer journey and this is of good sense they leave one organ they have to enter the blood circuit they then have to exit and they have to establish in another place secondary tumor so lot of changes happen there from mechanical point of view the idea is that elasticity stiffness might be a marker a bio free label marker for diagnostics investigating cell mechanics helps to understand cell alteration so is not only diagnostics but you understand mechanism so in general is accepted that cancer cells are softers and non-neoplastic cells the question is is it always true because otherwise there are problems with diagnostics so what we did we did a comparison between AFM experiments and optical tweezers experiments about this optical part the procedure Suleiman will explain to you but we had and we have complementary values for forces bigger forces for AFM low forces for optical tweezers stiffness and also the loading rate so how fast you push on your sample we studied three cell lines which are characterized by different neoplastic level so HBL normal, almost normal cells no plastic low metastatic potential high metastatic potential I do not enter into detail of these things but anyway when you do the experiments you should understand also if you damage the cell and where you should measure because as with our body a cell does not have the same stiffness everywhere above the nucleus near the leading gauge and so on so here is just a picture of the AFM image and optical tweezers optical microscopy and then we did experiments first with AFM and we measured from the nucleus toward the edge of the cell to understand how the young modulus goes and it is decreasing and then we confirm this also with optical tweezers with optical tweezers we are not able to measure so fast so we did three measurements one above the cell one intermediate and one at the leading gauge the good news is that the two procedures to techniques gave similar results in which terms in terms of trend that is that for single isolated cells both optical tweezers and AFM indicated that the most aggressive is the softest but if you observe here the absolute values are three order of magnitude difference so AFM, kilopascal optical tweezers Pascal why? because the regimes of techniques are different so one conclusion here important conclusion is when you say this is a stiffness of a cell you should say you measured with AFM with what you measured you where you measured and so on so in our opinion a good in this paper we stated that measuring above the nucleus is the best then another point is not isolated rare cases when they are isolated they are connected with other cells they sit on something so how much the cell stiffness is influenced by cell-cell contact and cell substrate contact so in our recent paper we studied first cell-cell contact and we had the surprise that MDAs most aggressive cells get stiffer when in contact being similar to HBL and MCF so you built a building forgetting all the conditions so the idea is not always the most aggressive cells as a stiffer as a softer if I go too fast please here then one tries to understand from this mechanical properties which is a structure which elements in the cell induce this property we tried so this is a confocal image of acting once slice and the nucleus for connected and not connected cells frankly speaking we could not find the conclusion for this but in the same year there is a group in fact they published in ACS Nano we published lower impact showing here is the important information showing that for the stiffer cells green now is microtubules and acting is red so you do not have red on the top of the fiber on the top of the cell so this means that if you have less acting it is softer or less softer in principle it is softer no ok, mechanotransduction what means mechanotransduction you apply a force you stimulate mechanically the cells because the cells they do not react only to biochemical signals they react also to mechanical signals things that they touch each other then it depends on what cells, what tissues and so on but definitely there is an effect so one of the experiments that we tried we tried because we were inspired by the work of Michael Schietz who is in Singapore has an institute of mechanobiology there was this let us coat the bead with fibronectin which is characteristic to the extracellular matrix of the cell and test what happens when we press with different strength on the cell what happens with the cell after so the idea you have fibronectin which reacts to intra membrane integrin protein and then these binds to vinculin and vinculin binds finally to actin so the idea was ok, here we exert a pressure and we want to see through vinculin which was transfected we see the reaction of the cell and what we notice we notice that these are three beads and on these three beads we apply different power for trapping the strength and after 20 minutes we observe that the intensity of the fluorescent signal indicating the accumulation of the vinculin is more or less proportional to the force applied so the cell reacts at different forces, at the changes of the forces we thought that some more sophisticated way to stress the cells what you see here is a hella cell going under the cage so we built a cage of beads then we come with the cell and then we stress the cell somehow this is a too sophisticated configuration for what we could do in biology we did it with diffractive optical elements and projecting the modulator and here you see how it better how it works so the cage is this you have one bead on the top the other level you have three the other you have other three and the cell comes here and then you go with the structure on top a nice example recent example which I like quite a lot is mechanotransduction to calcium signal calcium signaling so here what it is about it is force applied to a bead on the cell study the calcium influx into the cell and the calcium release from the endoplasmatic reticulum of the cell so calcium is essential in all the cells in neurons is more than essential so understanding how the calcium regulates for instance the cytoskeleton rearrangement is very important and here what they used they developed they are there is a group of 20 persons so from different institutes they are very strong in developing threat probes biosensors with which you can follow the calcium the binding of the calcium so is a common model in much detail to avoid that I bother you so the idea for so using the threat is I think Alberto already explained to you is that you have biosensor and you use in which you have a donor and you have an acceptor and you excite only the donor and when you have an activation of the molecule so it reconfigures and the donor and the acceptor get close each other you have the emission from the acceptor ok for instance here what you see you see the activation of the calcium or the activity of the calcium when force it is applied with beads coated with different proteins so is fibronectin which interacts with the membranes proteins and it is I think is BSA which does not interact so in red here you see that when a force it is applied on such a bead the cell answers so the activity calcium influx while here does not I just want to show you so the force was applied and the flesh you saw was a calcium release then you can build they build wonderful experiments when they blocked because why is the calcium goes inside because you have ion channels so ion channels they are like mechanical valves something should come to open them and usually that is if it is mechanical it is tension of the membrane the tension of the membrane is induced by force by placing the bead and pooling so ok so we arrived at biochemical stimulation induced by coated beads manipulated in contact to the cell or filled lipozons optically manipulated in the vicinity of the cell and photolyzed and then the effect on the cell is observed by optical microscopy techniques in the same platform before I begin I give you an example not from our group but a very beautiful example by Kress at all what they did they incorporated the micro beads polymer micro beads biodegradable micro beads in which they incorporated chemo-attractant molecules and chemo-repellent molecules in terms of the cell chemo-attractant is to tell come here, come here, repellent and is just example to show you what they did on neutrophil cells why? this was in my opinion very smart and they published very well because neutrophil cells are extremely motile they move by their role they should move because they should capture they should go around so what you see here is a bead this is a cell and the bead is moving and these are scissors and this is real time I mean it's two minutes three minutes experiment and this is chemo-attractant and this is with two beads chemo-repellent the cell tries to go there but there is a release of repellent molecules from the sources saying no no here you cannot come very nice fortunately for neurons it's much more difficult to apply this and to control the rate of this so what we are doing as I said is we are studying neurons young neurons and the gross cones and also how so we try to mimic a neuron or the signal from another neuron to the neurons that we study and then you have two neurons which in two hours speak to each other and you see that thylopodia come touch so they investigate each other and you see I don't say that these are synapses because it's not enough to shake the hands to make a business it should be something more but the idea is that we take the second neuron aside and we want to study the effect of one molecule that is released by neurons on the other so our goal actually is to create physiological inspired experimental conditions mimicking one of the two neurons because in biology I think 98 percent the experiments are done like this you have the cells cultured you want to change the chemical conditions what you do you pipet some microliter of and you soon have a constant distribution of molecules because of the diffusion but in general biology and we all we live because of gradients think of gradients of any type gradients space gradients time gradients chemical gradients so gradients are fundamental and they are fundamental also in biology so one way to stimulate so a bit better is micro pipet based assay that I told you about smart is with cage molecules where you have so you have the molecules cage and we switch them we free them by light and this is very popular very useful in neurobiology unfortunately there are very few molecules that can be cage so our approach is was introduced initially not for neurons by sun and chew and we applied to neurons so we fill liposomes with the molecules we wash the solutions and we take the liposomes we put them in ambient near the cells we trap one liposome we bring it close to sorry I have the explanation for this so I don't want to waste time for the functionalized beads they are commercially available very cheap functionalized with carboxylic group which allow you to attach any type of protein on them fill liposomes they are like this we put them inside you see how it works this is in contact if you want to break if you have liposomes you have to break you will pulse laser to deliver the molecules so one experiment one first experiment was with BDNF break derived neurotrophic factor this is probably you have heard about neurotrophic growth factor they are important because they are kind of nutrient let's say for cells for neuronal cells they are particularly important because it has a role in the synapses long term synapses so what we did because it was a question open question was this can one bead coated with this protein stimulate, trigger the BDNF signal and the answer was yes and we started this first that we trigger the receptor and this means that you see the phosphorylation then that you have the change of the calcium signal and then that you have a translocation in the nucleus for the C-force I do not show you all of this more information is in this paper I show you just the calcium so how we do this is a neuron these are the dendrites we first place a control this is important to place a control to see if you induce some effect and we look at the calcium and the calcium remains constant over 10 minutes then we add another dendrite the stimulus the BDNF bead and we see that the calcium level increases not only in the cell body this is the cell body but also in the dendrite then we make checks with two beads BSA to to be sure that there is not a mechanical effect but it is the chemical effect ok, so the liposomes as vectors also here they are quite a lot of freedom degrees we use this version so because we put hydrophilic compound inside preparation is quite simple so they are spherical vesicles from 50 nanometer to 50 micrometer phospholipid bilayer membrane aquas core this is also a configuration interesting to use with lipopholic compounds so you insert the molecules in between the two layers of lipids but we use just this one number that I think that it is important is that a liposom of one micrometer diameter filled with one nanomolar solution contains in average one molecule so we like to work with one micrometer because we see them well concentration nanomolar you can get so you can make experiments quite interesting so in this case we create a gradient because we have the liposom here here is a point on the cell and here it is represented how the concentration goes is a function of time function of distance from the source so you see that in time we have a saturation so after a while we get the same concentration but in space we have quite not very sharp but quite concentration so an example of this is a project that we had with we have still a group which professor Lenjame who is studying is studying prpc this is cellular prion protein which is a very important protein because it has a lot of functions which are not yet discovered the versions and isomorph of this is a scarpe prion that you might have heard might be as origin of a lot of neurodegenerative diseases but let us take the good guys a prpc it is not well known how it works and it is everywhere a bit like I don't know the name in English but a bit like Prezzemolo Italians parcel a bit like parcel so prpc in the cells is a bit like parcel in at least in Italian yeah to say that it is everywhere thank you so what we studied and we found this molecule works like this protein works like a guidance molecule so it guides the neurons in the case of neurons to grow also it was interesting that we found that they need so the prpc without sorry the prpc is also its receptor so if we used mice knock out for prpc so without prpc in the membrane the effect disappeared which means that you need to have the same molecule on the membrane to induce the effect then full length and so on and so far now here you will see the guidance so here is a liposome here is a gross cone of the neuron and you will see the photolysis of the liposomes and then the gross cone and the dendrite goes towards the source in 80 minutes or so you see that it elongates grows and comes towards the source ok so we run the same game before with guidance molecules because we were interested to see how many molecules are necessary to create an effect and one important conclusion is that this that around 5 nitrogen molecules on a gross cone are enough to initiate attraction but much more sema 3a which is repellent molecules are necessary for repulsion I do not run this movies maybe this because this is the collapse so you see the photolysis and then the collapse of the gross cone because it is the same offering sending the signal ok this is the last one so here we complicated because by now you saw only morphology effects from our group I showed the calcium which was calcium signal but not from our group we had only morphological so the idea in this project was to study signal transduction so chemical molecule and then which element from the chain of the pathway of the signal is activated and in these cases we were interested about GTPases and mainly about CDC42 so just to show you that this is a simplified diagram of the pathway of the signaling so the point in biology is that you never have ok independent signal propagation so these are the nodes of information membrane that this comes here, this comes here no, very soon you have this and this goes here and this goes here and it is this is a complication because you have to separate and to study some of them here in short what you have is configured the guidance cues and the receptors so guidance cues come from outside here is the membrane here are the receptors and then the pathway and then you arrive to the effector and the F-actin polymerization which makes the changes in the morphology no in this element is CDC42 which binds to PAC3 and triggers so sends the signals to effector so what we did is said ok, we stimulate the cell locally and then we look into the cell with threat probes designed to let us know CDC42 and PAC3 bind so we work with two types of intermolecular and intermolecular probes intermolecular means that you have the donor attached on one molecule on CDC42 and you have the acector attached to another and they are free so floating around and this is experimentally a problem because you do not have stoichiometry 1 to 1 so you do not know how many of that and how many of the other you have when they bind and then you have threat so this other prob, intermolecular you have both the donor and the acector on the same chain and you look only as a configuration change so this is more it is easier and more rigorous experimental the point is that intermolecular biosensors are usually much more toxic so you have to find a compromise technical compromise between rigurosity and between damaging basically the cell so now we work with both but at the beginning we tried first with intermolecular and we went back to intermolecular ok, the setup is something like this it is a optical microscope inverted where we have optical tweezers we have the laser to break the liposomes 355 pulse laser we have the fluorescence source and then we have an auto split with which we we project on our camera we project the fluorescence from the donor and from the acector because this is fundamental for interpreting threat ok, this is the neuroblastoma the gross cone we place the bead here after 30 seconds the trap is switched off the bead goes on and we follow for 15 minutes and we see the retraction of the cell you see that it retracted from white to red and this is the activity of cdc 42 spontaneous so without any stimulation and the high intensity reflects higher activity and this is much more moved it is stimulated skip this because I am already out of time and it is also some of details now about correlations I just mention that very interesting experiments can be done even with much simpler things instead of using beads you use biological particles we run quite interesting experiments with extracellular vesicles which are vesicles released by cells diseased so they send information to other cells also this conclusions so we can measure forces we can apply forces we can handle vectors carrying activity molecules to stimulate local cells and we have the advantage that everything is compatible with optical microscopy with imaging which means that we can see what we manipulate and manipulate what we see so here thanks to many people who collaborated and that's it thank you very much for your talk


Hello, everybody. Welcome back to another AI video. This is a quick one. I want to show you Hyper AI. This is a free tool. It's free as of March 22, 2024. They may change that in the future. Free AI tool that allows you to create video from text or from an image. You can animate images, a lot of different things, completely free. Let's go first step here. You'll see here I'm on the website. Links in the description below. Let's go try for free. When you do that, I'm logging in directly if you haven't because I've already logged in. If you haven't logged in, well, no big deal. Just go ahead and sign up with Google or Discord. Once you log in, you have a few options. I'll show you just a few of their videos. This one here is just this really cool looking Lego movie style. Looks pretty good to me. Let's click out of that one. And then here's another cool one here that I like. And then really, really interestingly, on the right side, you can see here that the actual prompt that was used to create this video is included as well as the seed and the ID number. So if you're familiar with mid-journey, you'll know that you can use the seed and the ID numbers to kind of repeat and create this type of style. So anyways, there you go. These are just a few really cool examples. The first thing you want to do, however, if you want to create a video from text, go ahead, click up here. Just click on create video with text HD. It's kind of like create with text prompt. I don't know why they have them both here, but this is the one you want. Click on that. And then down at the bottom, you're going to see a pop up and you're going to see a whole bunch of different options pop up. Just keep in mind that whatever you describe in this little prompt here, we'll get, you'll get. So I'm going to go English Bulldog, reading a book. And then you know what? I'm going to use one of their pre-built styles. I'm going to use the cyberpunk one. And when I clicked on that, you'll see here it says cyberpunk style, high tech city, mechanical enhancement, yada, yada, yada. I'm going to go ahead and click create. However, keep in mind that if this isn't correct, you can delete and add in as you see fit. I'm going to go ahead and click on create. It's going to take you into the creation mode screen, which is this one here. And you'll see here that it's queued up. And while I do that, you will notice here on the right side that I have gone ahead and made some previous generations. So if I just show you a couple of them here, you can see here, if I click play here, I've got this Bulldog reading a book. And of course, this is also cyberpunk-ish. Limited motion in this one here. This is an old retro style one. So that's actually really good. Looks like it's from the 60s. Yeah, it's a 60 style. So there you go, these are just a few examples. But anyways, I'm going to come back when this is done, show you what we got, then I'll show you how to animate your images as well. All right, welcome back here. So here's what we got here. We got this kind of slowish Bulldog doing his thing, reading a book now. This is a very slow motion video by default. Reading is not an extremely animated activity, but of course you can go ahead and add in as you see fit. If you want to change that up, make it more active go nuts. If you want to download this, all you need to do is hover over the down arrow with the bracket underneath it and presto, it will download it, you can share it as well. And then interestingly, if you want to regenerate this, let's say you like the video, but you maybe want to change its style and you want to re-roll it basically like an RPG, you can go ahead and click on regenerate. It will redo it with the same input if you want to adjust it. So if you want to vary the prompt, like this one here, this instead of reading a book, maybe you'd be running on a beach because that would maybe be a little bit more active, have a little more motion in it. I would just click on that and I hit enter and it goes ahead and does that. Now, while it is doing this, I want to show you one more thing here. Up at the top here, when I click on explore, you're also going to see animate your image HD. I'm going to click on this and this is interesting because now at the bottom, I can upload an image. So if I have an image that I want to use, let's say this one here of this cyberpunk lady enjoying a beverage, I can now go ahead and animate this specific image, this image I made in mid-journey and now I want it to be animated. So I'm just going to go, you know, attractive cyberpunk, I don't know, just whatever I want it to say. You know, drinking an energy drink. Okay, sure, something like that. And then I can just go ahead and click on create and what's happening now is it's going to create both that video that I'm regenerating, you know, but instead of the bulldog, you know, reading a book, it's going to be running on a beach and it's going to create that animated image. I'll come back when they're both done, then it's, then we'll wrap it up. All right, welcome back. So here we go. Here is the attractive females having a drink here. I'll just hit play and you'll see here, there's not a whole lot of motion in it, but there's a little bit.  And then to be honest, the English bulldog running on a beach cyberpunk. I mean, this is not that good. So I would redo this and I also did redo it. And this is the second one I got. And I mean, it looks like the dog's flying backwards. So again, not perfect, but keep in mind free tool. So all that cost you is your time. Last thing I want to point out, however is up at the top that you can not yet, but one day you're going to be able to extend your video. They are very limited right now. I believe two or three seconds is the maximum, but eventually this is going to come. And while this is not Sora or Sora AI, you know what, at least this has shipped. So it's got that going in its favor. It's completely free. Give it a try. Link's in the description below. Thanks for watching.