 We will continue our activity with hands-on activities. It is now time to have an experimental session on optical tweezers. We have manipulation of micron-sized particles and discussion with students. It is a nice experiment prepared by Professor Dan Kozrok. And the tutor, Dr. Muhammad Sulayman Yusafzai. I have to repeat for several times. It's like a music. So it is dedicated to everybody. First, you will have the explanation and then please follow the indication of the professor and the tutor. And take care of the indication. Do not touch the experimental setup. Enjoy your time for experiment. Please, Professor. Thank you. Okay, so I will try to be short with explanations because I know that you are interested more in the experimental part. I see that many of you are tired and probably after the conference reception or the workshop or whatever, winter college session. So what we have here, we have an optical tweezers, modular setup from Torlabs. And it is composed mainly by a laser, a fiber laser at 975 that is directed towards this objective onto the sample, which is put on a sample holder and where we insert microbeads in water and we will try. Then we have the illumination. We want to see also what we are doing. And we have the illumination part through the lead, which through a condenser, which is lower numerical aperture, illuminates the sample and the image is formed on this camera. Then the second module, this is an optical trapping and manipulation module with which you can trap, move the particle and observe. The second module is for force detection and this is composed by, I start from the end, by a position sensing detector which receives through a lens here which images the back focal plane of the condenser onto the sensor. So it receives the pattern which is the interference pattern obtained from the scattered light from the bead plus the light not scattered taken from the samples through this condenser. Then you will see also other components. For instance, this one is nanopiedzo, which allows to move in three directions with nanometric precision in order to calibrate. Because to detect position, you need first to calibrate the detector, which means that you need to move the sample with a precise value and see which is a signal on your PSD. And well, then there are the controllers and we will discuss a bit more after. So this is the scheme as I explained to you now. So I can add that you need a beam expander. This is important because you need to fulfill the pupil, the entrance pupil of the objective in order to use all the numerical aperture and to create the gradient. You can trap. If you send a laser beam which is not expanded, it's not enlarged, you do not trap, you just push the particles. Okay, one important feature of this kit is that comes from its modular design and is the fact that you can easily implement other imaging or spectroscopy stuff on it. So for instance, you can insert fluorescence, you can insert Raman spectroscopy because mainly basically what you have, you have an infinite pass here. So as we speak here, here is a detector, here is a camera, sorry, here is a laser. So here we can insert through beam splitters, different input for fluorescence, for spectroscopy Raman, for laser dissection, whatever. So in this sense is modular. This setup was developed at MIT by the group of Professor Lang and you can find more information about how it works, about different applications in this paper in American Journal of Physics, optical tweezers for undergraduates. And then it was taken, it was produced by Torlabs. By chance I know the story because one of my students at that time, 2005, 2006 was at MIT and he was very impressed because he noticed besides the very precise optical tweezers for measurement setup, 30 setups and he told me, Dan, here people play with setup, each student has the possibility to work and knowing our difficulties here. In fact, they were working on this type of setup. So it was like a practice and feedback from students before producing it. So I'm not making publicity of Torlabs, I'm just saying a bit of the story. And as characteristics, so you will find and we can discuss more. So we will find on these materials that I will upload the server and we can discuss more. So the trapping module, as I said, has a laser, has a trapping objective, high numerical aperture, oil immersion, but it is not the only option. It can be water, whatever, the important thing is to be high numerical aperture, has a depth of focus about one micron, the spot size about 0.6, I speak for the wavelengths that we use, the working distance 230 micrometer, which is not big but it is enough, while the condenser and has a good transmission also for infrared. If you build your own, when you buy the objective, take care about the transmission curve because usually the objective microscopes are designed for visible light. So to be optimized between 400 and 800 and then the performance might fall down quite fast after this. Okay, the stage has, besides the nano, it has also, because the nano positioner allows you good precision, but this one, because it's low cost, has a limited range, 20 by 20 by 20 microns, but it costs only 5000 euro or so. Then to move first the sample, the stage, there are the micrometer and there are the differential micrometers, which allow you to move with one micrometer precision mechanically, so in all the directions, and then there is a usual micrometer with which you move faster. Then the position sensing and force module, it might be quadrant photo position detector or in our case today we have a position sensing detector and the trap stiffness is in the range as we discussed in the presentation. We use our code to process, but from MATLAB they have also nice software and given software for processing the data, it costs something like 2000 euro or so. This is the last slide, I think. I have to tell you a bit some of the story. Now this setup is an ICTP setup, which was bought with funding from ICTP and the SPIE. It includes, besides these two modules, also beam steering with galvanometric mirrors that we have, but we didn't bring them here because they are very sensitive and in a demo is difficult to present. The story behind this is, if I am not wrong, like this. So in 2003 we began trapping, building our optical tweezers and we were working also with ICTP step PhD program. Soon we had many requests to visit and work with the only setup that we had there and it was very difficult to run and to have visitors and so to make your work. So we were beginning to think about one possibility to have a setup dedicated for practice with students coming through ICTP. Initially we sought with Joe to build a simple setup transportable for demo. Unfortunately we hadn't money. I built one and that workshop in Ghana actually was that setup in a box. My friend Francesco Di Fatto was bringing and installing there. My friend and also my ex-student. Then it was produced at Torlabs. Money could, funding could come up and we succeeded to have the optical tweezers from Torlabs. Then we did some demos around so we call it optical tweezers kid because we bring it with the cars there and there and there. And we can make demos but what is important also is to transmit information and that it cannot be transmitted and trained in two hours or so just showing. So we had also PhD students, master visitors working on it and mainly this was within ICTP programs. So I would mention Fatou Mdoye who recently defended the PhD thesis in Senegal. I mentioned with pleasure Sulayman who will guide you for the experiment who did the PhD with the University of Trieste Nanotechnology Program. And the last year so after PhD he had one year ICTP drill and he will leave to the United States from April. Then for short visits, relatively short visits there were Jose Suarez Vargas, Humberto Cabrera and Alireza Moradi. I hope that I didn't forget no one. And of course I had always the collaboration of Joe Niemela. We didn't speak about nanoparticles. We will try to make metallic nanoparticles to we will try to trap them here. I remember that with Alireza Moradi we does not work. Okay, I tell you the story. It's a pity because why it's not working? Can you try to make it work? We try to trap nanoparticles and just to trap them against the wall as Tatiana Aleva showed to you. That you need the third dimension because otherwise you just run the video. Otherwise you push the nanoparticles. And what we observed? We observed that even if we worked with infrared light and the particles were iron oxide at a moment and the absorption is low by these nanoparticles. We observed that at a moment since many particles are aggregated together the cross section increased and we got the thermal effect. But the thermal effect not in the sense of the thermal lens but we got the bubble because the temperature increased very fast there. Can you run? Please? Running? Yes. Okay, so you see actually not single nanoparticles because the resolution of the microscope is not such. Nanoparticles are, I think here, certain nanometers. Somewhere in the center you see that more or less in the center of the screen you will see accumulating nanoparticles and some of them we can stop them against the cover slip. The configuration is like this. And at a moment there are more nanoparticles. It's a bit long this movie, no? A bit boring. But keep your eyes on there because at the moment it will come the nice part. So this is just to show what you can do with nanoparticles with 2D trapping in a relatively simple configuration. And I take the advantage also here to tell about a simple experiment using optical tweezers as a tractor. So for instance at the moment we had to study 2 types of liposomes functionalized with molecules which get together but they bind. Only if the concentration of calcium, I think it was, it is over a limit. And with optical tweezers we could do this experiment very simple. We use optical tweezers as an attractor. We put together the liposomes of both types, no? And then we switch off the laser. Here's the molecules, you see now the bubble. And this behaves since it is air inside, this behaves like a convex, like divergent lens. And it attracts also other particles. No, no, it's okay. We lose that. And what you see, not very well here, other particles coming, being attracted to go further. Yeah, and then soon you have another and so on. So this would be nice to be described. Okay, I finished here. I pass the mic to Suleiman and I let you follow this. Okay, so these are just the beads, three micron silica beads. I just to check that they are focused or not. So we will go to our... So we are using this optical tweezers setup for force measurements. Force measurements, they are the same like when you indent a cell, a cancer cell or a normal cell with an AFM. So we wanted to do the same kind of stuff with optical tweezers. So normally we call it optical tweezers because it's a mechanical tweezers, so we replace it just with a focused laser and a bead trapped. And we call it optical tweezers because it applies piconewton forces. So it's like a ball or an object connected in all directions with springs. So we can utilize this property and if we displace this bead from the normal position, there is an attraction because of the gradient forces and we can use this as to calculate forces, which is proportional to the displacement from the center. So this is exactly the setup. But here we play with the final term, which is this is the interference pattern of the bead on the quadrant photodiode or on a position detector. And we play with this interference pattern to calibrate the movement of the bead. And from that movement we then calculate the forces. So the simple thing is if you have a quadrant photodiode or a position detector, you have three outputs, delta x or x, y and some signal, which we use for axial indentation. So you just take the position when you calibrate it or not. There are two methods to calibrate because if you don't calibrate it, it's just an attire so you can just play or a video game. If you calibrate it, you can apply it on the cells and in the bio and wherever you want. So the first method, there are mainly or basically three, but we use two equipartition theorem because when a bead is straight, it's moving with a Brownian motion and it has like three degree of freedoms. And each degree has KBT over 2. So this is the freedom, the thermal freedom of the bead. So we utilize this property KBT over divided by the variance or the Brownian motion. So we can calibrate this. We can take out the information about the stiffness. Then next one is power spectrum density in which we just trace the bead just like we will be doing this. And then you plot the density function of this variations. And then you fit with the Lorenzian, which is this one in red. And then you have equations. So this is the plateau and this is the corner frequency. These are important and we will try to extract this information here. So there are so many vibrations and then you can use these equations to find the stiffness and sensitivity. So the sensitivity is important because sensitivity defines the movement of a bead and how you detected how much voltage it generates. So this is the simple stuff just for calibration. We will try it just to, we will play with this. The optical tweezer indentation, as I mentioned in AFM, there you have a cantilever to indent the cells. But here you have a tray bead and then you can indent it also just by looking into the change in interference of the bead axially. But if you look at the forces, as Dan mentioned earlier, that up to 50, like piconewton, you come with a cantilever, but after that the vibrations in a fluid, they are like, you cannot measure so precisely. So then we can come to the optical tweezer setup and with this we can measure from 10 to below 0.001 piconewton. So this is quite good and we use this just to establish a setup to, it's like to acknowledge the AFM or in the lower force regime. So we studied elastic modulus of the cells, of breast cancer cells. How we did it, we move the cell down. So we, and meanwhile we record, we move the stage up and the bead is straight. So it indents the cell also, it displaces depending on the softness of the cell and we record a plot like this. So this is the bead trace which we record with QPD and this is the signal. And since the setup is calibrated, so we can identify that, okay, how much when the bead moves a bit inside and then the cell, so we call this indentation and from these simple calculation we can extract that how much is the indentation and how much is the bead displacement because the interference pattern on the QPD changes and how much is the stage displacement which is SD. We know this because we give a particular signal to the system and then just using these you can find out force which is the bead displacement because this is important and then the stiffness is definitely its KB2 or variance of the position and then using Hertz model. So we, because we have everything over here, we have indentation, we have the radius of the bead. So we just normalize this equation and we plot F against indentation. This is, we linearize and we plot and from this loop we define the elastic modulus. So this is the, in red, this is the stage motion, this is the bead motion which we record with the QPD and the black one is indentation at which depth the bead, the cell is indented and then we plot it and from this linearized region we identify the elastic modulus for each cells. Each cell, so we repeat this for many cells, then we take just, we apply statistic and then we can categorize that which cell is softer and which is more stiffer or stiffer and which cells because we know that the soft, the cancer cells when they are isolated, they are softer but when they are in contact, so we did such a kind of studies. So now I will finish here and we will start just playing with the setup. So a few things. So this is just the CCD. So I will shine a laser from here which will come like this with a dichroic and here is, so if I say because I make it like white, so it's the space between, because there are two core slips, so the space is between the two core slips like 50 micrometer or so. So I'll just insert a few ml, a microliters and I did it so you can see the beads and then we will focus the laser, you will see somewhere here and then you will see that the beads are coming toward the tray. So it's like a balance of gradient and scattering like that forces. So let's try because we have beads and now we will turn on the laser. I will keep the power very low so you will see the beads not so fixed because it's IR and I can't see. If I want to see to align it, then I use some cards to see that it's the laser is here and I have to wear the goggles but for now it's cover so it's safe. So from here I'm giving a 50 mW of power. So there is somewhere to see so I have to move the stage up and down. If you see like a blink then tell me that where it is because here the monitor is not so well resolved. You see something? No, because it's... So here the tray is somewhere here so I will... because there is a spot. If I increase the power, so this is the focus laser focus. No, because I turn on the light because to have a dark background to see the laser reflection. So here is now the... This is the focus now you can see. So I will turn on the light again to shine like this. I'll put a pointer over here. So let's try that our laser can trip or not. So you can see. So this was the... Even if I close it... Okay. If I move a bit... So... If I reduce the power... So I... Now... If I do it like this... So if my setup is calibrated I can use this bead to indent a cell or to attach a DNA theatre to pull it or to attach it with the membrane of the cell and then pull the theatres. So I can do whatever I want in a biological medium in a physiological medium. And now if... If we find a stuck bead... This is now the bead and these are like the plots. Now I am collecting the data from this position sensor detector. So let me check again. If I run I am using this dog and then for five seconds I am using... So this is the trace. These are the traces of this bead, the Brownian motion. When it move like this the interference pattern on this QPD. It's changing and I am recording this. Even so I saved it somewhere. This one. So these are just the thermal vibrations. I convert them. I took the densities of these and this the plot is like this. It should be like Lorenzian but it's not. But normally it comes like this and here the plateau starts. It's not because we are using low power then the vibration... It should be isolated from any vibrations. But again if it is like this we can click... We can select the... So we plot our Lorenzian fit according to our data. And if we say okay this Lorenzian it shouldn't be because it should come like this and in a bound state it should be like this. Straight. But it's not because... This is just a demonstration. Also because there are low frequencies so there we have a bit of everything because the power spectrum shows you the density from low to high frequencies and you do not see a plateau because the contribution of the low frequencies is important. If you say okay this is yes this is the correct behavior of the bead inside the tray. Then you say yes and then you can proceed further. If you say yes then for the X and then for the Y it's not good but we say yes and then for the Z so in three directions it gives you the power spectrum yes so it shows you like it calculates the sensitivities like 0.5 it's millivolt per nanometer it means if a bead in a tray move one nanometer so it will generate a voltage of 0.5 millivolt so these are the sensitivities and these are the stiffnesses which you say that this is 0.3 piconewton per nanometer though these values are not correct but it should be like this that how much is the sensitivity and how much is your tray stiffness so we calibrated like this and then we have if we know that this is this has a particular stiffness then we just indent the cell or interact this bead just measure the displacement of that bead and we have a tool to calculate the forces now we can trade even more if I increase the power to like 100 millivolt so now we have two beads yes let's switch off the laser and the beads even if I move the stage so I don't have so because the laser is off if I turn on the laser you can see they are attracted and they are so it's like this so now if I move I will have these two beads so because we are looking from the top one bead is trapped the other bead comes like this and if we are trapping so they will be on the same if we trap another one maybe it will not so the bead which is free not willing to come so I lose this one okay so here the window is like because the two beads so if I am traveling the bead which is down so it's it is obstructed by some dust and then I lose it so the optical tweezer which we apply forces is the same if you move against like this and you displace the bead and then you can apply piconewton forces less than tin piconewton if I turn on another plot it's like now we are going to move the stage yes okay okay let me have what I am doing now because I am moving the stage up and down so it's like a kind of indentation you can see if I record it so now so it's the same even if you move up and down or you move it laterally you will have with any particular and with this you can identify the viscosity of your fluid just you move and when you lose the bead it's the escape velocity and we are now trying also to find the different fluids with different velocities so if I increase the amplitude I increase the frequency so I will have different escape velocities depending on the viscosity of the fluid so if I say okay I am using like 6 to 7 micron so if there is some obstacle over here so it will just pinch or just strike that particular region so all this is like now we are playing with the kind of video game so yeah so you can join you can tape or so it's like the house is open now so you can ask question and Suleiman sorry if I can so let us organize like this if you have questions now and then if you are curious about the components and so but when we go there we switch off the laser to avoid any so to be to respect the rules for laser safety we have 6 goggles but it is impossible that we organize everything so if you have questions now or later please thank you as I have noticed in this slide for measuring the elasticity of the cell he used the Hertz model so I don't know if I am remembering right but the Hertz model is assuming both mediums both surfaces definitely to be elastic but the cell surface can be classified as visco elastic I should thank you for the question you are right the point is this so the cell is mostly viscos is not elastic but or almost all the results with which you confront your results in AFM are built on a Hertz model so we know that these measurements let us say do not correspond to reality that you should investigate in fact you get information about viscosity and you can use it but if you want to confront with the results with others you have but you are completely right I agree in fact this is an advantage I would say of optical tweezers and working at low loading rate but at least in this case also when we used the Hertz and model I put my hands on the head because I said my god what we are going to say here about the stiffness this is a definition but is a definition about whether or not it is a wrong measurement any question? there are no any questions we should organize for I don't know in small groups we have two rules that if there are two people one behind the other you cannot see so this makes part of the good sense and the other rule is about to see the table so with a bit of patience and who is curious to see the components and discuss you can approach to the table please even if we want to turn on the laser so it's good to take the goggles for a few minutes because we have time so yeah you can take the goggles so we can turn on then when we will turn on so no no it's off now yes but we will turn it on yes and who I don't need it I'm married so it's normally if even if I turn on okay but I'll use like okay 30 is quite so I can trade so the laser come like this if we want to detect the laser so put a detector over here yes it's infrared so you have to take your goggles the trapping is this one the laser goes like this and here is the objective and it's somewhere there this is the we inject the fluid and this like because I made it just you know I just use the white tape the double tape to yes to be visible to no but the power is quite 30 milliwatts so it cuts more than 70% so it's like so this is one laser is here it's 50 this is not 75 the 51 here and I have one here so the beam is expanded four times and then yes to fill up all this and it goes here it trips and the interference pattern is generated on this QPD by this this is the lens and this is now the PSD that not the quadrant this is just the position sensor yeah but if you are using the axial studies just like an FM you need a QPD and we took the some signal normalize it and then we can use it for using axial studies the laser is coming here here and then so it's four times because this no because it's not damaging the cells it's transparent to the cells we are using biological media so it's yeah none is waiting to be you can touch you can so infrared because we are using the biological media so yes so we are not heating up it transparent yes particularly 450 we are using 100 here the exit power is one thing but when it comes here it like more than 60% it yeah you lose all the so at the end you need 20 or 50 milliwatt but since it's focused but the infrared yeah transparent so you have very low absorbance no now we have these are the silicon beads yes the silicon beads are they are they bigger in diameter than the beam or not which one yes yes these are three micron and the beam no it's like one micron yeah yes no it depends which particle you can trap these particle three in one the one done was using yeah bigger to actually we didn't bring because we have there the two mirrors like this so you can create different shapes glass particles yeah yes it's not the power it's the numerical aperture which matters yeah even if I can reduce it to 10 we can trap there it's 1.24 yeah yes yes it's like yes yes yes but yeah but yes yes yes even with the because we have a special because we have heating here and we have a special holder for this because we need to no because if we are working with cells they should be at 37 degrees otherwise if you are major at room temperature the stiffness changes and to work for one hour at least in a safe yeah yes yes it's no no no here we heat it we just fabricate this because we need a heat the other one it was not yes but here we here is the remote couple this one and this is the heater this strip yes so we measure quite many times just the temperature were here so it heats this chamber all yes here so this is metallic so it heats yes it heats up this region and not to heat this we have here a glass not to heat the whole assembly to damage it yes can find so yeah where is that liquid where you have the so it's like this yeah the double tape I will remove in a very rough way I'll take one we are not using this I'll put it like this because I took I think many yes I did it like this I'll turn off this I'll remove this since so there is a small channel ok so I I have to add because it's an oil immersion objective these are the gold nanoparticles let's try this maybe yes yeah if I turn it on just to see this is the focus yes though it's not yeah no like circles yeah it should be but it's like a polarized when you are using a polarized light so this split in like four leaves so you ask this I just drop it suck it with yes four laps yeah what is the size of the particles these are like yeah 15 nanometer because I don't know but these are agglomerates it's not you see because these are gold nanoparticles they are pushed away yes catering because so we will push it up and we will go to follow it here somewhere the upper surface is here so it pushes up and we will follow it against the upper cover slip if you have a 3D trapping for the gold nanoparticles you need to yes then we have now gold nanoparticles just to gold nanoparticles just to follow it yes to follow it to the on the screen for those who are there they are gold nanoparticles now now you might see if we switch off the light they are not particles they are agglomerates aggregates yes no these are these are just this is X this is Y and this is to move it up and down no no when I trip the particles so the particle move just by particle striking the so it's just move with the brownian motion it moves with the brownian motion but the trip bring it back to the center so it's like this and we have a lot like this for this yes for everyone if you are in a medium and because you cannot reduce the thermal vibrations KBT energy shall always be there no for everyone in a medium there are there should be one degree of freedom which is KBT so if it in 3D it should be 3 times KBT this is because to have a visibility because you cannot see with the CCD then single nanoparticle for that you need to collaborate your position detector because anything which enters the trip it creates a kind of fluctuation so you can then play with it even when you attach a DNA with a bead even the same bead and you have another trip or pipe it to pull it you just see that you are stretching something this bead is fluctuating but you don't see the DNA so no anyone else want to come nanoparticles no they are they are not but in any case they are trapped along the optical axis yes many particles actually we don't have a shaker we are here to yes individual particles nothing because it's a 15 nanometer so you can see a bow 200 or 300 but not below because it's a diffraction limit for this one yes because with visible light you can see that yes but for that you need detector suppose PSD which create an interference pattern it's a position detector or quadrant photo detector so if you have a trip and something enters even with nanometer because your voltage fluctuations are for in nanometer milli volt per nanometer so if something enters so it means that your interference pattern will fluctuate and you can calibrate that one to trip so you can say that okay if you have a uniform kind of so if you have suppose 15 nanometer gold and you trip that and you will have a particular kind of interference pattern and corresponding voltage if you trip to it will change and so so you can say that okay I have one two it's the same like thermal lens you know the previous so you just if you have one layer of graphene your thermal lens effect is different when you add more the effect changes so here then you have one then two three because they will be along the the direction the axial direction so the interference pattern changes so if you calibrate that you can say that I trip one particle two particle because one particle like 20 nanometer and this you can see only with the TM or same so so it's not possible with the optics just CCD to see if that was possible then we can see DNA like but it's stretching so it's the same when I have I attach a DNA with two beads suppose yes so I will trip one or one is suppose stuck so I will just pull the same in presentation or with a pipette a micron pipette so you have a trip and then you are pulling the pipette and they will so you can actually it's like the coils it's yes unwinding so with every push you have a signal so it's like going up yes like yes and the same like when you have an octomycin motor which moves so you have a bead which is straight and the other is on the on the bead with that motor protein and it goes like this on the octin filament so in each step it pulls there the bead so you can detect that position because it moves you give energy and it moves so you have these steps so they then calculate their okay for each step in the muscle cell will I do like this so for how much energy is needed and how much actually motors they contribute to our muscle contraction and all this stuff so it's very sophisticated but it's possible to design a group in US they are doing the same thing two beads and they attach DNA and then they stretch you see yes with proteins and so you can play it depends on that what you want what facilities you have yeah if you can handle so it's that's why they call single molecule because they want to know from the very basic that what is happening with a single particle tray you can study like not as a bulk for a single single particle you can study the surface plasmons like and one you can shine that one particle you can yes yes so with a surface no no it will be there because but not so much that you can you just started so it should be so you can touch this that it has some current or not you can touch this to play with this because maybe not done is not here so you can touch if you go to the toilets maybe they have a complete kit and they have a price yeah so I forgot I'm sorry this is just to move this from our computer through this so if I move this like this so it's I'm moving it with nanometers precision electronically if I move this this is in micrometers and also in and if I move this this is one micron one micron and this is an mm yes yes x y and z so when we are working with the cells so when we trap a beat then we use this to come to the cell in above the cell because if we touch this so that effect because it's a sudden push so it's last for long so if you move micron by micron you are not affecting the you are not giving any vibrations yes x x y and z so so this is okay this is in the so normally this is the cell and we have read about and we and then we the move the stage up and down only because when you are working with so it's closed now but you can see that there is so if you put your eye here and you cannot see but you will damage it yes you see it's and then it here and here so it comes also here because it it collects and then it project on here this is a laser yes it's 10 20 or something like this yes only one so what Dan said the counter propagating so if I take this laser like this put it here and another laser like this so they will be going like this like this so in between they can trap a particle but it is like now single beam so what it's it's coming like this focus is like this so at the center because the change in momentum the net force is towards the center so that's why we have for a single beam we have it's not like one is like a magnetic levitation when you push against but it's like it go like this because the change in momentum so it trips at this focus this is yes it's simple it's yes yes yes yes now it's to heat when we are working with cells we need a temperature of 37 degree yeah not die but we at least one hour we have we are safe because we do not yeah no because just yeah just you just need to have a wider kind of yes yeah so we can use nanometer from here yeah and these are this is no position detector PSD take the goggles because the laser is on lens here in which you align the position how do you align that exactly you just watch this point in alloying or whatever here is also a pointer you see okay so it should be in the middle lens as long as this is on the center yes right now because something is trapped if the trap is off then it has to be on the center yes so if not again but this yes it's and these are for the laser power yeah it's a diode laser diode laser which way please it's a 10 yeah this is no no this is the camera CCD the laser is there the fiber like this so it comes like this here is a dry acrylic so it comes like this this if you want to yeah this no with this with 100x yeah yeah collecting yeah yes yes yes this is the problem this is the yeah if you want to collect all the light which is catered by the or which exit from the this objective and to yes just so you need to put it close and to have a high numerical aperture yes but then if you reduce this then you cannot put because this sample when you are working with a cell it's like in a petri dish or on a cover slip so you need to put it then you don't have space to put your samples it should be like this so no no this is you yes yes you can take don't take it but no it's it okay with a fluorescent tell me if you want I don't know what is next