 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 a reviewer, take care, read well what you receive. 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. This is a last slide, a photo of him from the lab and his technician, Zijecic, and him in the lab. 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