 I think that due to all things that we can do today with a microscope, we can change this sentence in this microscopium extraordinarium nominarellibuit. And since the microscope is sort of star, if you watch to the sky, you can see these two constellations related to Galilei that are microscopium and telescopium, I don't know if you have ever seen them. And since with the microscope, mainly we produce images, a lot of other data, but mainly images, I like to bring to your attention this sentence by this catcher, Yogi Berra, that is very interesting because since he was a catcher, he really had to observe in a very sharp way and pay great attention to the small dynamics of this guy that was launched in the ball in order to understand where to move. And so I think that we are within this framework with the microscope, biological cells on other objects we are interested in. So today I will try to discuss with you about fluorescence, I don't know which is the background, but I know that background is not homogeneous, so I will try to give some data about basic of fluorescence and application and so on. So I'm sorry for those of you that are killed in fluorescence. But as first start, I think that we have this start that is related to the fact that with the optical microscope, we can see what is invisible not only in terms of dimension, but also in terms of interaction with electromagnetic wave we are using to probe matter. This problem of having biological objects, the one we are interested in transparent was solved by Zernick as you know and you had lessons about face contrast so you can get some contrast from objects that are normally transparent and later introducing some markers in the sample that can be coupled with this kind of approach that are fluorescent. And then later you can mix approaches and you can move to making the invisible visible in colors. And colors are not only for beauty, colors are because colors related to fluorescent molecules and biological objects have a very strict relationship with function and structures we are interested in biological systems. Now we are working, when you know about the electromagnetic space, we are working in this very small region. I have a specific reason for working in this region. I come from Genoa and colors of my favorite football team are red and blue. And this is the reason why I focus my research in the visible light. So from red and blue and when you have interactions, you have absorption, you can have scattering, you touched all these points and absorption related to absorption, we will see something about biological systems and then we will move to fluorescence. In this region that is from red to blue you have some biological systems that absorb light and so that in case can produce contrast that are in the near infrared like amoglobin, lipids and water. And then in the ultraviolet, most important I think is DNA and then you have some elements in the ultraviolet that not only have absorbance but also produce fluorescence, this can be of interest. Of course we like not from the microscopy point of view but from our living state point of view that biological macromolecules we have inside do not absorb light otherwise our temperature under the sunlight would increase too much. And you know that within this or probably you know that within this window you want to avoid shining light on your system in the ultraviolet because you have 220 and 260 absorption of protein and DNA and so in case you shine light there you can change their structure and so the functioning. And on the other side if you move to the near infrared you have some water absorption windows and then you have some collagen absorbing there but as you see here also you have signal that region. Amoglobin is very important and we don't want to touch this when we plan to go into living system also using the microscope because we can change oxygenation and we can change conditions there and we don't want to do this. So the main reason we are using the optical microscope is because I've not asked you to become solid, to be physically sectioned, to be metallized and to be injected with electrons in order to have some contrast. I simply can't follow what's going on using light. So these are the windows that we will try not to touch too much. And using the microscope and so this was an observation using again a contrast there is something that does not induce too many transformations within the sample. You heard about the optical microscope so you know about all this stuff. You had the beautiful lessons last week I've seen all the slides there. And now let me go into the optical microscopy scenario for the topic of today that is fluorescence. So the idea here is that within this scenario that is rather complicated or complex I don't know. You have two main categories and today we are mainly interested to this one related to probes approach. And I hope that I will be able to convince you that when we talk today and I don't know if in the appropriate way or not but we use this term super resolution in microscopy we are mainly focused on preparation of the sample on the probes and their properties more than in the optics needed for producing super resolved images or images that we call super resolved. So we will try to move in this domain. So fluorescence I don't know if you have been here at the Capella Sistina. But if you have seen this painting you cannot escape that this color looks different from all the others. It looks a little bit brighter or something attracting you more than other colors. Because molecules making this color have also another property. When you shine visible light you can see this. When you shine ultraviolet you can see fluorescence from there. When you use fluorescence and you use fluorescence with a biological cell you take advantage of a lot of science made in order to have biochemically speaking something that is very specific for things you are interested in within the cell or in cell aggregates on in cell aggregated in tissues and organs. You can be really very specific. And so you know or most of you probably know that fluorescence is the light emitted within a finite duration subsequent to the absorption of electromagnetic energy. And now we are shining energy in the visible and situation is not so complicated. Usually what we do is to excite our molecules so to shine to send energy using light so to shine light at the appropriate energy to bring the molecule to an excited state. And then within these time scales waiting for the decay. And you can have decay or not. There are some parameters regarding the fluorescent molecules ruling this. But usually what we exploit is excitation of the molecule some weight in time and then back emitting a signal within this time scale that is 10 minus 9 10 minus 7 second down to the ground state. And then you can start again this process. Like you when one is talking too much you stop listening but then you can get a coffee and you can start again. And with fluorescent molecules sometime after 40,000 for example cycle for fluorescence they stop emitting forever. Some of them can restart but most of them stop forever. We will see about this property later. Why fluorescence? Side of David Jameson that was a student in the Weber lab, a Gregorio Weber lab, it's pretty. So this is one reason. But the other reasons are that with fluorescent probes you can be biochemically specific for certain macromolecules. But you can also have your molecule so the fluorescent part of your molecule that can be sensitive to these conditions. And so you can use this not only for understanding where a molecule is in the cell but what is going on in the environment. Very flexible. And so you can use this for molecular structure and dynamics, cell organization and function. You can work in living animals. We will see or most of you probably already know or some of you don't know but you can do this. And of course you can work on engineering surfaces. Again these are more or less the key points in the excitation. Usually so what you do is bringing the molecule here and then you have your molecule spending some time at this level. Losing some energy and then relaxing back from the lowest of the excited state down to the ground state. Now we are now depicting the molecule as something that works in a frozen condition is not. Because we are working room temperature. So it's really a mess what's going on here. And this is the reason why you have probabilities in excitation and probabilities in emission. It's something that is not blocked in one condition. And so you have to consider the probability of exciting your molecule. So you have what we call the excitation spectrum. That in some cases overlay with the absorption spectrum. And if you consider a pure substance you have to consider that not only that the frozen spectrum is invariant. But also that does not matter which is the wavelength you are using for exciting. When the molecule is in the excited state the probability budget of a relaxation is the same. You don't have to care if you bring the molecule by temperature by a chemical effect or by a single photon excitation to photon or three photon excitation. When the molecule is there the probability of emission is the same for that molecule. And this guy is the one responsible of the description simplified that we use as simplified description of the absorption and the emission process. And for some pure molecules you have this mirror effect in absorption and emission so you can predict also the emission from the absorption. If you go to some molecules that we use in biology we have for tryptophan we have the possibility of absorption and we have an emission that is in the blue. Then you have other molecules and including DNA that you can label with the specific molecules like this one that allows you to have an absorption and a consequent emission in the visible. This to say that you can use light to deliver energy to a molecule that has the property of releasing energy releasing photons. And this is the mechanism we are interested in. But now if we have a look to the fluorescence decay. Starting when you start exciting the molecule you can also have an analogy with the radioactive decay. There is a lifetime associated and we will discuss about this lifetime of the process. We will discuss about this because temporal window for releasing photon from a fluorescent molecule is really strongly dependent by the environment conditions. And this can be good or not but for most of the modern application of microscopy this is extremely good. When we think about fluorescence and microscopy I think that most of us most of you but most of us including myself think about intensity. And a specific spectral window but we have so many things that we can collect in fluorescence. And that we can use for learning something from the molecules emitting that probably you cannot imagine or you imagine but you didn't use. Now there is a quantity that is quantum yield that tells you the efficiency of your molecule in converting the in releasing back the energy. And so as initial point you can think that your molecule receives energy. And then one is in the excited state. As most of you can release the extra energy I don't know becoming red or crying. So becoming red you see a signal crying maybe you listen sound. And the molecule can go to the ground state releasing some temperature so without any radiative decay or releasing photons. These balance is the balance that you use these two possibilities these two rays of the activation of the molecules are the one of interest for defining the quantum yield. The molecule you would like having a quantum yield one but is not. Let's assume that now you have only two possibilities so the fluorescence and the not fluorescent decay. This is the quantum yield and we associate the lifetime to this ratio that depends on the possibility of the molecule of going to the ground state. This is relevant because let's assume that you have a molecule that is working in I would like to say normal conditions but not perturbed conditions. And so this molecule only has two possibilities one is. Going back without any photon released and going down the releasing photons. And then you can define a lifetime of the senior release by the photon that is the lifetime. If conditions around change so this balance changes so Ki changes what happens is that tau changes. And so even if your molecule is emitting in a very same spectral region let's say in the green lifetime of the process is different. And if you're able to measure this quantity you can map lifetime point by point. And you can find that your molecules your fluorescent molecules even if they emit the very same wavelength let's say green. They have a different lifetime telling you a story that is related to the environment. This is an additional information that you have and that you can use and you can refer here to the population of the excited molecules and to the variation of the this population of excited molecules. In order to find the relationship. That tells you which is the meaning of the lifetime that you can find in this. Final relationship that is the number. For us and molecules of photons released from the fluorescent molecules divided by the initial number in time during time is changing with this behavior and we call this lifetime. One over E is where we find it now can we measure this why not so what we need to do is to shine to send our energy to the system and to start collecting. If we start collecting and this rule is true what we collect is something like this. And so we can measure lifetime. There is a problem in this kind of approach that is the kind of approach that people was using at the very beginning for lifetime measurements. Is that in order to have a good statistics. You need to collect a large number of photos and in case you want to build an image. That is I don't know 500 by 500 points. It takes a long time and when it takes a long time and environmental condition changes. Is possible that there is something that is changing in time and tells you reports some changes in the lifetime. And so you can have a wrong view of what is going on on the overall system. We will see also tomorrow that we have solutions for removing this kind of program. However, this is very important and. You can have measurements of lifetime in different ranges as you can see here and today we are really able to measure changes in the range of nanosecond. You can perform this experiment in solution so the environment is all the same and you have an overall average condition in order to check which is the. Lifetime of your molecule under conditions that you are able to control. Then you move into the cell and into the cell, you are not able to control anything. That's is a free running there for your molecules. So again, you can measure in this way, sorry for this. And you have this case, so you have the possibility of reporting point by point or measurement by measurement, which is the lifetime of your molecule too long here. Maybe the rest, sorry for this. I want to go to another point. It is always the very same story. I I would not comment or discuss now what's going on. If you have two lifetimes, so now we are talking about the molecule and we are considering only one constant of decay. You are in condition that you have multiple lifetimes. Of course, you can distinguish them, you can study them, you can separate them. Situation is more complicated, but you can manage it. I was just telling you that lifetime is something that you can measure and that is relevant for understanding what's going on with your molecule. Is there another method than exciting and counting in time for measuring lifetime? Yes, you can also use a frequency method that works on the modulation of the light in time. And in this case, what you measure is something that you use to measure that is the amplitude of your signal and the phase. So what you can do is to shine your excitation, to collect the emission and to quantify the phase shift. The phase shift between excitation and emission, that is the temporal window of this phenomenon, can tell you something about the lifetime. It's not so precise as the measurements in time, but it's fast. And since it's modulated, signal to noise ratio is better. So then you will have this slide, but we can demonstrate this and we can demonstrate the relationship between phase and the lifetime. So these are the two main ways that you have for measuring lifetime. Sorry for this, I want to just show you what really means having this possibility in terms of imaging. This is your intensity image for this sample. It doesn't matter the sample from my point of view now, however, is a salient protozoa and we were interested to understand what's going on in cilia in terms of distribution of molecules. And but if you measure the lifetime of the very same molecules that are producing fluorescence here, you see this distribution. This means that they are in a different environment and they have different relationship with the sample. And since you have different colors or different contrasts, you can have a better contrast in terms of image, not an increase of resolution, but a better contrast. But now, since we are talking about fluorescence, there is one aspect that is the one that was mentioned at the very beginning. That is the permanent loss of signal from a fluorescent molecule. This is photo bleaching. Then you can have blinking, you can have other effects. But when we talk about photo bleaching, according also to a textbook written by an Italian scientist in photo chemistry, this is both Tirolli. Photo bleaching is when you have lost completely any possibility of getting a signal in terms of photon from your fluorescent molecule. Of course, this happens because the molecule changes structure in time. So now, since you already discussed about microscopy, you can assume that you have a gel, three-dimensional gel, fluorescent. So this gel is three-dimensional. The view that you have here is z-axis, that is the axis of propagation of light, and x or y. And now I'm shining light in the gel in a very small region, so I put my focus on my lens in a very small region in the three-dimensional object. I shine light until I consume all the fluorescent molecules, so until they photo bleach all. If I do this, and I realize this when I see black here, if I have a look to my gel in the three-dimensional space, I realize that I bleached also the molecules where I was not interested in and they were out of the focus of my lens. This is expected, or that can be unexpected when you perform your experiment. You wanted to probe something here, and then probably you decide we will see later, and you have seen in the past days, we will move in another optical plane. But when we move in another optical plane, and we have this effect, we can get the wrong view of what's going on. Because, for example, let's assume that we have a big cell. You have some speculation about the fact that DNA is distributed in some way within this cell. You shine light in one focal region, and you see the signal, and you say, ah, the cell is alive. And then you move to another plane, and you don't see any signal from DNA, and you say, ah, this is a cancer cell. It's not. It's simply because the signal photobleached before you moved there. So this process is an interesting process, but you have to take care when you attack your system in terms of fluorescence. So what's going on here is that you have your molecule that is normally performing this game, but then in time, the signal is fading, and you have the bleaching of the signal. Again, here is the same. And these are the processing bringing the molecule outside from the scheme of getting an excitation and releasing a green, a red, or a blue photon for your image. Now, another bad point related to photobleaching is due to the fact that different molecules have different behaviors. And so it's possible that you have photobleaching in different temporal windows, molecule by molecule. This is another reason for taking care of this phenomenon. And we will see later when we will discuss about single molecules what you can learn from photobleaching, and that is true that you can expect for an exponential decay of your signal in time while exciting using appropriate photons for bringing the molecule to the excited state. But this is really a key point also in the development we will see tomorrow of the super resolved methods. You cannot escape from considering this problem of photobleaching there. Since behavior of the molecule is dependent on the environment, if you change the environment, so if you reduce the possibility of oxidization of your molecule, you can change photobleaching conditions. This is the reason why when you look to your fluorescent molecule, you can consider if you are looking your molecule in the cytosol or in a fixation that is using a certain substance like, I don't know, prolung. This is a way for reducing the production of molecular oxygen. So what happens is that you have less photobleaching than in other cases. But again, photobleaching is really a critical point when you are performing fluorescence measurements. Fluorescence again. Now, you heard about, I assume that you had some lessons about the optical side, from the optical side to photon excitation. In this case, we are interested only on one aspect now, later on the other aspect about the instrument, but now about the fluorescent molecule. You have the nice possibility of bringing, following the Mariega-Permeyer prediction, of having your molecule in the excited state without delivering the appropriate energy. So let's assume that your molecule needs 10 in terms of arbitrary units to go to the excited state. You can deliver your energy 1 plus 9, 2 plus 8, 5 plus 5. 5 plus 5 is more practical. So using the very same light source instead of using two different light sources. So let's assume that we use 5 plus 5 now. And you can bring your molecule in this way to the excited state. We will see that this game of ability, because temporal windows of this event are not exactly the temporal windows that we are able to manage in terms of waiting time or in terms of probability of the event. And I will be back on this relationship later. But just telling you that you have a temporal window that is very short and that you have a probability of the event that is related to several parameters that we will discuss later for the fluorescent molecule and later for the instrument. Let's make this possibility of exciting a molecule using two photons instead of using one photon of the appropriate energy. Let me say that this means moving from green to red for producing images. The overall effect you have, but I will be back on this immediately, that you have is that when you want to excite molecules, this was the discussion we had before, just a few minutes before, you want to excite molecules here. You shine the appropriate light with appropriate energy. So what you do is to excite all the molecules along the pathway and you have higher density where you have higher density of photon. Nothing strange. When you move to two photon excitation, since the event is not a common event and requires a lot of requirements to be primed in terms of excitation. But the final effect is that you have excitation of fluorescence or fluorescence only for a very small region. So you can really be selective. Just for fun, but we call this two photon excitation. You can think that this is the Winnie-Pooh philosophy in case. But despite the name, it's not a game made by using two photons. You need a multitude of photons that go together in a cooperative way to the very same target. The name is two photon, but it's not two photon that. So you really need this. And you also need something special related to the temporal window. So you need to have the exact time of this event. Let's assume that here in this room we organize a coffee break and we give you the time for the coffee break. And we ask all of you coming into this room to send energy to a bowl that is here on the ground and to provide an energy that is mg divided by 2 with h. So half of the energy needed to bring the bowl on the table. If you play this game, you come here, you play this game and observer in this room will see probably always a bowl at the half of the 8th from the ground on the table. But now if we tell you that there is a coffee break and we tell you exactly at what time is the coffee break since you know that if you arrive two minutes late, nothing is left. You will arrive all in time. You will provide, all of you will provide energy to this bowl and you increase the probability when you try to provide this energy to have the bowl at half of the 8th and so being in the bowl, the table. So you really need something telling you something about timing of this. Increasing this probability with density. And if we have a look to what happened in literature, now we focus tomorrow, we will focus on these two years but now you will see that you had this prediction. In the 60s you will see in the next slide there was the demonstration of this effect and Pauline Sheppard in 77 demonstrated the possibility of collecting a fluorescent signal exploiting this nonlinear interaction of light with a matter, so a two-photonous citation. I assume that he saw the signal and then the sample disappeared or something like that because of the high energy used in the focal region but later using different lasers and a different approach there was the first demonstration of the possibility of using this for biological system and in particular for neurons by Winfried Denk. So again this was the story of sin and this is part of the history related to photon and citation and this year is the year where you had the experimental observation due to the fact that you had the invention of the laser so a light source providing photons at a very high density. Now in terms of interaction with your sample and what we can get here from the photon and citation what is nice is that before we needed blue or ultraviolet well not ultraviolet but 360 that is moving to the ultraviolet region that is a dangerous region for the cell because you can induce structural changes. You needed this for priming for example fluorescence in the marker of DNA now you can use 5 plus 5 so 720, 720. From the point of view of the sample is better because red light is absorbed definitely less except for the window, water windows. In terms of the signal that you receive back nothing changes. You have fluorescence, you have excitation process primed so you have fluorescent molecules in the excited state and so the realization follows the very same rule of the single photon excitation. Now there was this calculation made by Denk's Voboda and in case in a bright sunlight you are in a boat under the sunlight and you want to experience a single photon event in terms of fluorescence your rate is more or less one second but if you want to experience a two photon absorption event we are talking about 10 million of years in Italy we had only one person able to wait all this time in the past he is still alive but we had only one so in case you don't have this temporal window you have to find a way for solving the problem and the only way for solving this problem for this event that is not very common is to increase the density of the photon so organizing a coffee break with a lot of people around the molecule you are interested in and so you are in this high photon density region and you have to work with time and space space you have the lens so the best is the lens because the highest density is the apex of your double cone no matter time you need to produce this two photon or n photon effect in a way that you are not changing due to the very high intensities that you need for having this observable window in terms of time that do not destroy your molecule so really you have to take care of the temporal aspect of your interaction this is not in scale of course but the solution found in the dank paper was having very short pulses high intensity 10 minus 15 second means that the molecule is not able to change structure in this window but you have since the density is very high you have a very high probability of bringing the molecule to the excited state then since fluorescence decays in 10 minus 9 seconds and this is the most common lifetime you have not only because I'm from Genoa or other guys are from Scotland so we don't want to waste too many photons we don't want to try to excite the molecule while the molecule is in the excited state because this perturbation is strong in that temporal window and so if you wait 10 minus 8 seconds you are sure that your molecules in case relax it back and so in case you prime fluorescence on molecules that are not in the excited state otherwise you can bring your molecule to the second excited state or you can break your molecule or destroy your molecule so this is the temporal window that people started using for two photon excitation so back to this relationship what we are interested in this relationship now is the relationship with the power you need or with the photons you need with your illumination source why we are interested in P and in the fact that you have this in this way because you are talking about two independent events that are the first and the second photon involved in and if you treat this as two independent event you have a consequence I will be back on this formula later again but you this already discussed about this temporal window the consequence you have is that you have a quadratic effect that we will see soon but if you assume that this is a cuvette containing fluorescent molecules and this is what you perform in terms of excitation using the appropriate energy 10 you see the double cone inside the cuvette when you move to photon excitation you have this event only in a very small region why? because in this very small region you have the highest probability of producing the event or the highest probability of having photons from your illumination source so again with one photon excitation you have this let's say sketch in terms of excitation and emission but when you have to photon excitation excitation here density is too low for generating in an observable temporal window emission so you don't have any emission except from this focal point now the consequence we are interested in are related to what's going on in terms of photo bleaching we started from photo bleaching now if you consider very same gel and now if you switch off all the molecules for photo bleaching in this region and if you have a look of what's going on in the adjacent region you discover that nothing happened and so now when you move your focus and you find information on other optical planes you are sure that apart from diffusion processes you are dealing with new molecules you are not consuming the molecules before observing them and now there is another aspect that is interesting in terms of fluorescent molecule and the sample and is related to cross-section I don't know, I never found papers related to the prediction of the two-photon cross-section maybe only for one specific molecule under very specific condition but modeling this is very complicated and you can only measure this and if you measure the cross-section under two-photon excitation you find a different behavior from the case of the single-molecular excitation you have a dice, you have more probabilities of making 10, 5 plus 5, 3 plus 7, whatever you want and the behavior you have can be very useful but first let's have another comment about this cross-section this cross-section cannot be predicted for a certain molecule but you can have an estimate a numerical estimate now don't consider this part consider only the number you start from 10 minus 16 that here you know what is it's a real cross-section in geometrically speaking now if you start having a look to 10 minus 16 this tells you that working in fluorescence is nice for all the reasons we were trying to discuss before but it's not very efficient 10 minus 16 when you move to two-photon it's 10 minus 49 when you move to three-photon it's 10 minus 82 and so on this means that your process has really a very poor efficiency so exciting molecules, fluorescent molecules using two-photon is not the best solution for exciting molecule in terms of getting a signal but could be the key solution for exciting molecules when you don't want to deal with overall photo-bleaching when you want to penetrate deep in the sample and you're using red instead of blue and when you have some other aspects that are related to your question and your need in terms of interaction with the sample but in terms of fluorescence it's not the best way for producing fluorescence it's more inefficient than the conventional way but now the behavior you find if you measure cross-section of different fluorescent molecules here you have the cross-section in Gopro Maya is that cross-section is not a bell shape like in the case of single-photon excitation is something that is broad across the spectrum it's so broad that you have a lot of overlaps molecule by molecule this means something that you can use in a very effective way when you're performing your measurements if you need to excite different fluorescent molecules usually you need different laser lines or different, let's say, colors or different illumination wavelengths and you know that when you shine in matter you try to focus in the matter different wavelengths they have not the focus in the very same position and most of the time what you need from your multiple fluorescence is to understand if some molecules are interacting so you don't like this doubt that you have even if you have chromatic aberration correction all the correction that... and you can put everything in the right track after your measurement but you have that problem and you need three illumination sources that are fluctuating in a different way and that are delivering different energies but now when you move to two photon you can use one illumination source for priming fluorescence in different fluorescent molecules and then you collect your signal you have only one problem here and we will face with this problem immediately after these slides you need to be very sharp in spectral separation because now you're shining your excitation if in a certain region we have three molecules they will emit photon altogether your detector does not know anything about the wavelength usually about the wavelength of the photon and you need to find a good way for separating in terms of energy their contribution in order to be able to distinguish them so spectral separation is something that is a key word in two photon excitation so this is just an example with the very same wavelength you can get the three colors coming from different region from DNA from the cytoskeleton and from filaments and you have different collection of tables that are reporting about the wavelength ranges for two photon excitation here you have another surprise nice surprise that is that when you move to two photon excitation now having in mind a rule is not true but is practical I mean it's not true if you go to the formulas if you go to the real structure of the molecule is a rule of thumb some way the rule is that if I shine 720 it's like exciting 360 it's like exciting 360 but through my sample it's passing light at 720 only in the focal region I have an effect that is exactly like if I am shining in a very precise position 360 so I'm affecting a very small region just to give you an idea in terms of capacity of water this is 0.01 femtoliter this is the amount that you are in some way perturbing with your excitation considering the ultraviolet component and so you are able to excite molecules like NADH that on one side you can be excited in the ultraviolet on the other side these molecules are related to the metabolic activity so it's like I mean if you use ultraviolet and you check NADH you are perturbing a lot the system and at the very same time you are looking for the natural metabolic activity it's good but it's not the best it's like asking someone to sing and to hit him or her with a hammer the way he sings I think changes and this is what happens with the cell but in this case you have the possibility of using a perturbation that is a low level in terms of perturbation with the cell and so you can get this signal but now there is another effect if you are able now to excite this kind of intrinsic signal from the sample let's assume that you are using single photon excitation for the scheme we have shown before even if this signal auto fluorescence is poor it's distributed throughout for all the cell and so if you are able to prime this you have a lot of background with a very poor signal in the focal region but now when you are moving to two photon excitation you have a poor signal because auto fluorescence is not very bright but it's coming only from a very small region so background is strongly reduced and so if you shine a different focal position your excitation this is auto fluorescence coming from membranes sample you see from every region a good contrast image changing plane by plane without contamination of the background using auto fluorescence in case you are shining 360 here or something like that you would see a blob then you can use a pinhole and your confocal for reducing something but it's a blob in this case you can really discriminate plane by plane what's going on using this kind of excitation now point here is that your detector is old-fashioned movie cinema amateur is only looking for black and white movies and sorry that was light you have seen that colors are some way relevant you are interested in your technique color image and from the side of your sample if you collect only without any spectral discrimination the signal coming from different biological molecules you really don't know what you have there it's a little bit a mess it's better if you go to the pink Floyd side and so you are able to select colors and so the key now for fluorescence microscopy any method especially when using to photon excitation is the ability of separating spectral information this is what you can get from different fluorescent molecules information is interesting can be morphological one you can say something related to even if it's poor the contrast here about intensity distribution so can tell you something but maybe this information is better if you're able to produce these without artifacts an inappropriate way because this tells you not only something about morphology about about specificity of the molecule and in some cases about their function in the sample now you have different ways for selecting colors you can use colored glasses this is a good way this means that you are filtering your spectral information blocking light you are not interested in one problem you have when you are using these glasses is that if you are interested I don't know in the red signal it's true that this filter is killing all the other colors more or less if they are not too strong if they are not reflections for example from your excitation but it's also true that is not transmitting red color 100% and so you are also losing some signal it's a good method but some disadvantages if you go to your grandmother or grandfather house you can find something having prisms with lights and when the light enters in from the sunlight you have a really romantic and nice view and you can use this kind of element for spectrally separating light and then when you have separated light in terms of color and they are shining colors in different positions so you have the spectrum distributed in space you can decide where we have to place your photo detectors in order to collect the blue light the red light or the light you are interested in and so prism could be a good way for separating colors then you can have other separation methods for example a diffraction grating again with the diffraction grating you can separate colors and then you can collect them in different channels one problem I see here for the elements you have at your disposal today is that you are losing a lot of light when separating colors using a diffraction grating so it's not transmission 100% it's not the prism but the prism is better and so you have different solutions in different systems using this method for separating light just showing you some of them in different commercial systems sorry for this but so you can separate different colors and into different channels taking care about polarization in order to get a better signal you have different solutions but this tells you these are let's say new generation but made of 2005 from 2010 tells you how relevant is the ability of separating spectral components when you perform modern fluorescence or advanced fluorescence microscopy every time you have to consider this solution you have also to try to consider how much light you are losing while collecting different colors I'm showing this but also for all the others is the same more or less you have a chain usually what happens is that you receive light different wavelengths then you start selecting one and then other components are passing to another element and you are losing every time light and more light I will give you a number later light and more light for the spectral window you are interested in because every step you are not gaining photos you are losing photos this for example in terms of this time I like this method because you have a grating here you lose a lot of light here but the idea was nice because you have you have a grating that is on a Galvo mirror so this grating is moving fast and if you decode time so time is in some way encoding the spectral component time of deflection for any position you have a different color sent to the photomultiplier and in case your detector is not has some capacity effects in terms of when you change the voltage if you have a fast detector you can also change the sensitivity of the detector according to the different budget of photos at that wavelength because it's true that when you have your sample with a block sensitivity you can get lost you could have saturation for some wavelengths and the poor signal for other wavelengths these are stupid practical aspects but relevant when you have to say something about your sample and so today however we are able to collect real time colors and separated colors using using optical microscope and in this case the spectral separation of 8 colors from the very same sample at the very same simultaneously again fluorescence with fluorescence now you can you can have what I would call several dimensions or several possibilities you can have fluorescence lifetime measurements we will see later what about threat that is an advanced fluorescent method you can also have information about diffusion of your molecule in different ways that are fluorescence recovery after photobleaching and fluorescence correlation spectroscopy and then you can move to second single molecule detection and then you have another mechanism of contrast that is not related with fluorescence but is related to the way you are producing fluorescence when using for example to photon excitation and so we can talk about seven dimension you have optical sectioning confocal selective plane of illumination to photon and these are the methods for the three dimensions so you can have really three dimensional view of your sample but this is not the reason we are using the optical microscope well is one of the reasons because with the optical microscope we don't need to physically to make physical sections on our sample the real reason is that we are able to introduce the four dimension that is time so you can follow events time this is the advantage of using fluorescent and not visible light and so also for big sample you can follow development of biological systems from embryos to other stages it's something that you cannot get with any other instrument and when you use fluorescence especially in a special way you can really track what's going on your living system and so you can better understand not only a normal or a pathological state but you can also understand which is the effect of a pharmacological for example effect or a surgical effect in real time colors that tells you something about specificity of the molecule and function and when you use lifetime you can see as you have seen before you can distinguish objects using a different mechanism of contrast and then you can use something that comes for free when you perform to photon excitation since you are shining a very high intensity there you can appreciate the a signal called second harmonic generated signal and this signal so if you shine red you receive blue this signal is relevant because for example in this case this is the tail of a zebra fish this is a small fish now people was inserting fluorescent molecules and now there is one general comment about people working in fluorescence that now that we are moving to super resolution becomes more evident people working in fluorescence they trust on what they label so no way so when they see a green signal this green signal comes only from the macromolecules K57F that they have labeled it is not very often they have dubs on this sometime people try to find some biological fiducial in the sample that is the nucleus and so they tell you please can you give me an image of the nucleus in order to understand what I think I have labeled is where as to be but nothing more now there are some biological components like collagen or some other that exhibit second harmonic generation so they change properties of light in a way that you can detect and this is the case so in this case you can see the muscle fibers of the tail in purple and in green you can see what you have labeled in terms of fluorescence and we will see we can go to single molecule and to nanoscopy but now since we are talking about fluorescence we cannot escape from the fact that there is a new category of fluorescent molecules that are available these fluorescent molecules are also responsible for some for some reason of the development of super resolved fluorescent metals green fluorescent protein so like this name tells you their protein these means that biologists people in biochemistry they are able to induce the expression of this protein wherever they want so they can control a process expression of a protein in a biological system the other property is that they are green fluorescent so you can also see them as you know these three guys got an Nobel Prize in 2008 this guy for understanding a mechanism switching on fluorescence in a specific mechanism of calcium release due to a green fluorescent protein later discovered and isolated Martin Chalfi was the one with his wife Tule Azerig understanding that this protein can be could be expressed in any mammalian cell no limits in this Roger Chen understood not only this but understood that photophysics of the molecule could be also controlled and so you can imagine just as the simplest effect of this understanding that the emission color could be controlled changing only some element in the expression of this protein but what is relevant here is that for example Martin Chalfi wanted to understand the development of the neuronal system in the synobilis elegans work or the development of this protein and expression in time in living system is something that is you don't have to inject you don't have to stop the situation in injecting you can use this for following behavior or living systems while they're living so you can assume that you have an experiment with a tumor mass that is injected with green fluorescent proteins and you can see this big mass in terms of fluorescent in the mice and then you can start with the treatment and you can see this mass diminishing or increasing so you can really track what's going on in living systems but the big jump in terms of advanced fluorescence microscopy we had here was with the advent of photo switchable fluorescent molecules green fluorescent proteins this was the real jump both Mörner and Betzig when they had their own studies on single molecule detection they were in troubles because they were not able to control states but with photoattivatable you can control states of the molecules and if you can control states you can switch on some fluorescent molecules in a matter and then when this mother cell is dividing you can follow part of the of the mother cell propagating in daughter cell recognizing the fluorescent senior that can come only from the region of the mother cell that you have photoactivated you can really in a living system you can switch on something and then you can follow in the lifetime and so single molecule is relevant now with this green fluorescent protein and so tomorrow I will have some other topics to discuss with you that I'm not able to bring in this short time now but I want to touch this point now fluorescence the single fluorescent artificial fluorescent molecules green fluorescent molecules is relevant to try to understand something about the behavior of single fluorescent molecules because when we are dealing with a single molecule that is emitting we can improve our ability in localizing for example this molecule of this factor that is the number of photon emitted in case the protein is singing well the amount of sound sent but from a single molecule that is the only one talking to you time by time now there is one point how do we recognize single molecules because these molecules are small I mean so a green fluorescent protein has 27 kilodaltons this means that it is more or less occupies 5 nanometers in space depending on how is on the surface or in the sample and in case you are able to in case you have a single molecule on a cover slip and you follow the single molecule in time you can learn a lot in terms of photo bleaching behavior and some other properties of the molecule and you have different ways for getting images from single molecule you can have conventional illumination these are single molecules or could be single molecules or you can have a surface illumination I don't know if you talked about turf but you have an illumination occurring only at the surface or you can have some other mechanism reducing some background but more or less this is what you get now and this condition is the condition where the density of the single molecule is not very high now do you have any tool or any idea for understanding if you are dealing with a single molecule or not because when you have them in this case for example the structure is not resolved but you are always within your diffraction limit one experiment you can make is to have a very poor concentration of fluorescent molecule spread on a cover slip now if your experiment is so nano molar so in case your experiment is well done first of all there is no reason for the molecules of aggregating I mean in terms of surface charge they should not aggregate but this can happen but in general no reason and if your solution is at a very poor concentration and you put your molecules on a cover slip using some spinning strategy or whatever you want for putting them on the cover slip and now you measure the intensity of each spot you can have a distribution of intensities again if your experiment is well done it is possible that you have a majority population made by molecules having the poorest signal and then you have multiple of this signal the experiment is well done this would happen and in this case you can recognize on your cover slip or in your system by intensity where the single molecules are so are the only ones having one signature but now and so this is the real situation it's not exactly what we have seen there this is the experiment in this case with one micro molar and this is what you get and these are the categories you can have using fluorescence intensity but do you have another possibility for understanding that for example I don't know if it is one but this one is a single molecule what could you do for being sure because a single molecule will be the key word for the super resolution method also for learn something by single molecules and you want to be sure that you are dealing with a single molecule do you remember photo bleaching well if you shine light in time you have a signal from your single molecule and that this signal drops down in this way if you have more than seven molecules you cannot stop emitting photos all together and so you will have an exponential decay and if you want if you have a small amount of molecules so if your aggregate is an aggregate made by from one to ten molecules you can by counting steps you can count the number of molecules so using photo bleaching you can understand if you are dealing with a single molecule a small aggregate or a large aggregate this is a fluorescent property for those of you that are performing atomic force microscopy and force petroscopy probably you do the same so you pull and when you have a look to your force petroscopy curve you have steps telling you if you have one molecule or more than one molecule is the same because it is always something related or released by the system then you have another possibility that is very interesting that is used in polarization why not this is an oscillating dipole and so if you turn if you have a single molecule if you turn your polarizer and the molecule is immobile what happens is that you have a signal that can switch from zero to the maximum in 90 degrees if not it is an aggregate of molecules so you really have tools for understanding if you are dealing with single molecules or not and so now that you have this tool you can start studying properties of single molecules in order to use them for example in advanced fluorescence microscopy I will be back on this tomorrow but let me go until only the end of this part in past and then tomorrow I can discuss more about this if you agree this is the green fluorescent protein and this was a variant of this molecule the guy in tradition this was this molecule has a mutation here and what is relevant I will be back in case of this tomorrow not sure but I will what is relevant is that this molecule under two photon I will tell you the story under a single photon but under two photon you have this molecule having emission bleaching then if after bleaching you shine different energy to the molecule in this case a very sharp window that is 720 nanometer this molecule restores its fluorescence that time I don't remember which time but it was around 2004 we were thinking we had in our hands something for studying single molecules in an advanced way in cells the only problem we had in fact we stopped just for a while and too much and so others came with other molecules was that imagine we have to bring the cell expressing this molecule this green fluorescent protein we see the signal photobleach this was the critical point we had to photobleach all the sample and then restore fluorescence only in the region we were interested in and using a dose of energy able to restore fluorescence only in one molecule or few molecules time by time so this was the idea the fact that we had to photobleach all the sample we had too many discussions with biologists and so we stopped then you have in 2005 following the paper by Jennifer Lipikos Vards and George Patterson we decided to apply the very same to this photo-active GFP and here I can tell you the story it's shorter use your common GFP this is the mutated GFP you see from this excitation spectrum that if you shine light you don't have signal here but then if you photo-activate this GFP photo-activate means under single photon that using 4 or 5 you use the carboxylation in the protein this changes the structure and the excitation spectrum changes changes in this way and so your protein becomes visible but the great advantage here is that you start to dark and you go bright in that case we started bright we had to bring everything dark and then coming bright again one process more like when you fly one stop more so this was the situation for this molecule and if you have a look there was something that people started understanding with the wild type GFP but the effect was not so great as for the PA GFP so they stopped now what is relevant here is that you can create a population of labelled protein in a specific region the only point this is the reason why we decided to try this under 2 photon is that if you remember the graphical sketch about 2 photon and single photon if you use a single photon photo-activation you have a good probability of photo-activating molecules outside the region you are interested in and we were interested in a desired region so again this is what what happens when you perform this photo-activation using single photon and we decided to do this under 2 photon and this is what you can get so you can be specific in space you can really select the region here will be better in the next slide maybe here if this is the shape of your cell you can decide switching on only in volumes you are interested in you can measure your points per function and whatever you want the the region, the amount of proteins that you are able to switch on you can control this photo-activation and this is the reason why we were able to switch on in a precise way molecules within the nucleus that was the eastern H2B following what is going on in the daughter cells and so following an event in the daughter cells in case we were priming photo-activation using single photon we were not sure that the portion of DNA in the nucleus we wanted to check in the division was the one we were interested in and just to finish now this is my last slide this morning we decided to apply this also to this problem we wanted to understand something about trafficking in a cell so we had molecules in a vesicle and there were inhibitory or not of a process depending on the substances you released on a process of transportation of this molecule to the membrane two years more or less understanding this but then we were able to switch on the molecules only in a vesicle and so in the next stages of development of the cell when we were able to found to find them in the membrane the signal could come only from the one we switched on in the vesicle so in this case you were able to increase the what is giving you fluorescence because you have a protein that you can express wherever you want you can adapt to this protein a signal that is the green fluorescent protein you can switch on this when you need and with two photons you can be very sharp in the region where you switch on your molecule and so if you want if you now have a question related to a process in the cell from one region to the other you can use this for approaching then with fluorescence is not only for margaritas and you can get a lot of fluorescence here and this is what we think is the way for getting nanoscale information from your biological systems tomorrow maybe I will start with FRET and FRAP and then I will move to nanoscopy sorry for being so long this morning but I wanted since I didn't know the for everybody the same and tomorrow I will try to talk FRET FRAP and nanoscopy or starting with nanoscopy and then coming back to this topic maybe we can discuss which is the topic you prefer more if FRET or FRAP or nanoscopy if you have questions or we can get our coffee and we can discuss here I don't know what you do thank you very much