 Wave optics. We know that using ray is impossible to explain about the point spraying function, about the limit of diffraction. The best picture is using wave optics. In this case I will remember some important concepts. We have ray but also I intentionally add there the wave behavior of this case in which again we have here a point source. Let's suppose we have a point source, a bit, micrometer bit that is radiating light is fluorescent bit is emanating light in all directions and this bit is placed here in the center. In this case I show here that we can have also a bit a different displacement but the different will be only that the image instead to be here will be in other place. But let's we suppose that the this bit is here and that now we have an objective length here and after that we have a tube length. Let's we let we start with this picture this is the bit here and this bit as we know is emanating a spherical wave in all directions. But the objective is only able to capture this spherical wave coming till here okay. But if the bit is located at the focal point of this length we know that after the length we will have a parallel beam of light. This spherical wave will be converted to a plane wave. After that we know that for having an image we need a second length because a parallel beam of light has not any information about the image and a parallel beam of light with these tube lengths will be focused again and image will be created at the focus of this length. Then we have again this plane wave is converted to converging a spherical wave in a point. My question now is for a point object in a focal plane what is the distribution of light and near the image plane. What is this? Is it really a point? Not as we know. It's a intensity distribution there and this is called the point spread function of the microscope. In this case the difference is that I will speak all these things related with a microscope configuration. Let's go now I think Ana also teach about this or maybe we'll say something about the image. This is a very important concept about the huge wave lift approach that says each way from is the envelope okay we have here is the envelope of the wave lift each point on a way from acts as a independent source to generate wave lift for the next wave from each point is a secondary source of light. AB and CD are for example two wave fronts okay we'll use this principle now okay here also we have some important definitions each of the infinite points in the emerging wave from acts like a point source each point emits a wave flat all wave flat wave flat from the same wave from are mutually coherent very important that is they oscillate synchrony they are synchronized therefore the interference with each other in a predictable way and now let's we come from the two planes where the the plane wave is converted to converging a spherical wave because it's there where the image will be created and let's here we have these different wave flats of this way from and let's we start adding wave flats we start with using two wave flats only and then we will add three four five and all the wave flats and we will see what will happen there okay if we have only two wave flats intentionally I talk these two wave flats because they are these two wave flats that are far apart from the center of the optical axis axis and these are the last or the the the the maximum maximum angle that this or the object difference was able to capture and these wave flats will be related with the resolution of the microscope due to that we start with this and we were we are going to go down and let's see the interference phenomena we know that at the center of this because this is we have a symmetry there in this surface as Miguel said at the center this wave these two wave coming from these two wave flats are in phase they are mutually in phase and we will have their interference we will have a maximum of the intensity as we know but if we move from this point down or up for example here we will have a destructive interfering because we have half wave planes retardation and so on and then in the next point here we will have again the wave in phase and we will have again interference okay and so on let's we see here a better picture of this phenomena okay but what happened there really as we know the eyes or the detector reacts to the intensity in this case is a here is a some kind of representation of this wave left but we will draw here in this figure the interference pattern that produce these two wave flats okay but as I say you as I say you the maximum resolution is mainly related with these two rides here that here create this parallel being of light because this is a maximum angle that the objective is able to capture due to that these are very important way flat the string way flat as as we can see here as I say also yesterday the numerical aperture is related with this angle that the microscope object objective is able to capture we have high numerical aperture lower for working distance but we have a bigger angle bigger angle means as we know high numerical aperture and also higher resolution let's we see now at this picture what happened when the objective captured the spherical wave in the case unless we compare low numerical aperture and high numerical aperture here in the case of a high numerical aperture we have large larger plane and spherical wave of course it's very clear from the from the figures that this objective is able to capture larger part of the spherical wave it means also the plane wave here will be larger okay and after that the two planes is able also to focus a larger part of this spherical wave in the case of low low numerical aperture we have a smaller plane and spherical wave here this is what happened from the point of view of the way optics and of course a related also with the resolution and with the location of these two way flat in this case we have this way flat here and here are the string way flat they are separate away and in this case they are more closer and as they are more closer we will see that the resolution capability decrease let's we see now what happened here in this in this skin when we compare the the fry the interference pattern produced by a high numerical aperture and low numerical aperture taking into account what I said in the before slide what happened with these two way flats here that are far away what happened is that we have this condition gives a short fringe period and also give narrow fringes and in the other case we have long fringe period and white fringes as we can see here okay but let's we see also what happened with the point of view of the way flames when we have interference when we have interference using for example blue light at these way flames also we have this more period of light and in this case with larger way flame in this case around red 580 we have less period here of this way flame and the result of this is that when they interfere the face different will be different of course and we'll give you the possible the the results will be that we have also find a structure here similar to what we had using low numerical aperture or high numerical aperture and this is the case as we can see here in which we have give rise to a shorter fringe period and narrow fringes as was with low numerical aperture what does this mean it means that we have to use high numerical aperture and low a shorter way flames for having a higher resolution because the resolution is related with this finest structure there finest structure in this fringe higher resolution we have but let's we see what happened when we add more way the lights in this picture and let's we add another way flets we add the central way flets this is symmetric in this circle when we add these way flets we have two way flets at the ring of the length and one way flets at the center of the length three way flets are in phase at the center of the image plane this is the place the center because it's symmetric where all the way flets are in phase recorded intensity in this case graph bar the pattern turns into Iceland or fly at the image plane at the image plane center fringes are brighter because as I say all way flets are in phase and this is the picture of the of the result of adding more and more way flets but let's see now what happened when we have five way way flets when we have five okay tomorrow here again the brightest bang at the center of the image plane all way flets in phase again at the center of the image plane fringes a a and c constructed interference between way flets one and two and between four and five dimmer than a b okay of course over but dimmer fringes is still present the brightness as increase we add more way flets in this case we have nine and the result is that the brightness still increase the same phenomena and then we have the picture of all way flets there and the result is that all way flets are in phase at the center of the image plane light is concentrated in the middle of the image plane the main peak is bright brightest by far and other dimmer than the main peak intensity progressively decrease away from the center most of the light is within a cone at the at this at the center and the width of the main peak of course at the center set by the string pair of way flets at the ring of the length this means that the string way flets define the white of this of this central peak this is the picture we have here this is in the image plane and of course define the resolution what we obtain there as Miguel said and mathematically did is a the irides which is in this case the point a spread function of the microscope but let's see the effect of the numerical aperture on fringes in this case we have higher numerical aperture and lower numerical aperture is very clear here that why the separation between the way flets possible as a result a small small central peak and for the other case we have only natural separation between way flets and brought central peak let's see now what happened with different way flets when we have different way flets as I say before we have for the shorter way flets a denser area and of course narrow if we compare with the case the stream case for example of using red light and also in between there is a an average okay and this is the the point point spread function resource of the the result using different way flets okay let's now we see here this is another very clear picture again in the in the cross section the same irides but if you use if you use blue light this will be reduced in in size if you use a red light the iride irides spread it the same but using a what yes of course in c-direction we will see in x and y-direction but also we will derive now what happened in in set the direction sitting in in deep there we will see that the resolution is not the same there's some different we will derive this now and these are some relation important relation for the idea this I will not concentrate on this Miguel also did before but only to remember you some important concepts of a mathematical how we can do the mathematical calculation of these parameters and now the point spread function distribution near near the image plane in both direction as I say you what happened there as a as we know the resolution is related with this criteria in which we have 0.61 and the way flaring of light and the inverse of the numerical aperture in this case but what happened in deep in deep the resolution is different due to that you can have very good resolution in x and y and when playing but when you go in deep the resolution change more than in this plane in this case this is the relation of the resolution when we go in deep is the inverse of the numerical aperture and we have here an example this is the limit of resolution that we can have using blue light high numerical aperture obviative this is the example here using this way flaring and this kind of numerical aperture we have in x and y around 200 nanometers but in deep in set duration we have 861 more than this is not possible of course the light spread when you go in deep in set duration this is very clear but what happened with with the numerical aperture what is the influence on point spread function bigger effect on axial as I say than lateral spread as we can see here and we have one example here between low numerical aperture and we have lateral slightly larger spot and axial most longer spot narrower comb than the case of high numerical aperture and we have lateral compact spot as we can see here and in the axial long spot and white comb here is the resolution limit using this numerical different numerical aperture obviative and in depth it means this is better to use when we want to do a stack of image in depth is better to use a high numerical aperture obviative because we keep their better resolution than when we use this kind of a numerical aperture obviative okay now let's we see some examples what happened when we have a and let's we speak about the different criteria or definition about the resolution we have different criteria here the sparrow, rillet, half waff, half maximum but in microscopy the Riley criteria as Miguel said is the most important use criteria and is this criteria a state that the first minimum of the of the first irides is incomplete coincident with the maximum of the second irides and this is the definition of the Riley criteria and the resolution in the case of a microscopy there are other criteria of course but the Riley criteria till now is the best use criteria of course of course this we will see later that when using super resolution techniques another criteria can be used but we will speak later about this tomorrow and maybe also today and tomorrow let's see what happened with a Riley criteria in lateral and in depth we can see here when we look at the intensity how looks we know that what we can detect is intensity we at least can detect this minimum here and the difference between this maximum in in deep course and in the x and y plane this is from the point of view of the intensity but let's let's see now I want to speak about something that you just said now we are using here these are bits these are bits micrometer size bits with low numerical medium and high numerical but we are using the same magnification the same magnification of the microscope but with different resolution this is the point here is not possible to observe that we have many bits there it's not possible here we have so-so and he has very clear also the irides are very clear because we have a high numerical aperture as a as Miguel said this is a very good comparison answer to the question that way we can magnify this but we can have an image I don't know how how big but nothing there you can magnify also this but you will see all the details of the image and this is the idea to observe small details there the magnification is not important in this case you will see that the magnification will be important when we have to match the sampling theory with the resolution the diameter diameter yes of course of course yes it is all the one one micron this bit are around one micron is on the order of the wavelength I cannot say now in this case I don't know it's around but exactly I don't know doesn't matter for me where is the limit that you will see this this ring of course there is a limit and there is a relation between the diameter of the size of the bit and the wavelength of course they have to be close but we have to do I know this is the way but now it's not so important you are right it's okay now let's see what happened if we go down below this limit 200 nanometers and we have bits with less than this diameter if we have around around a bit around this diffraction limit around 200 nanometer less than this we have a bright spot we can see they are a spot what happened when we reduce this the size of the of the bit okay we can see they are similar we can observe maybe a little decrease less intensity of course and when we go ahead we can see that there is not any any change is similar the size the only difference is that this is less bright I will say that this is important when we use resolution techniques there is a so-called localization super resolution techniques in which when you have very small bit what they do is to localize the center of the bit and of course taking into account the brightness of this you can build an image but now we can see that there is no differing when we go below the resolution limit because the microscope is not able to resolve these small details it's the same for the for the microscope objective we have to go through super resolution techniques and I will show now very nice example maybe some of you don't know what what is there maybe you saw when I show my my presentation but I don't know what is this but if we enhance the resolution not the resolution we are using the same my microscope objective the question is about the sampling the frequency of sample sampling we enhance the paper the granular structure to say the the camera how in this case we increase the number of megapixels of this camera now we can identify that is a zebra there there is a zebra but details are not well defined even though that we use higher so high numerical aperture objective but we are not using the correct sample frequency we increase the sampling of this image now and go ahead and now we can see that here we have some lines but the zebra has this vertical or horizontal line also have yet you know but here is not clear then we have to increase the sampling the sampling rate then better better I will I will explain I will explain after that then we go ahead still we have some horizontal line here is not good okay now it's much better but the still here is not clear in this region we increase the quality of the of the of the detector oh now is better still okay the sampling the megapixel we are using more megapixel in the camera I will explain now why is it related with this increase yes it means that you can have very good resolution in your microscope but if your camera your detector doesn't match with this resolution and this is related what you ask now is important the the magnification you will see now one very nice example what happened we can identify the because it's a zebra and we know that the zebra only has line in this direction oh very good but what happened in science when we are studying cells we don't know what is there inside this will defy oh maybe this because this line is no no no no resolution to resolve these small details there okay what happened with the night nyquist the question or there the answer is the nyquist sampling theory what says that of course nyquist was working with acoustic wave okay and state that the sound must be sampled at least twice it is highest frequency in order to extract all the information from the bandwidth and accurate accurately represent the original acoustic energy for example human hair hairs frequency auto 20 kilohertz and cd sample rate is 41 44.1 kilohertz at least twice at least phone lines passes a frequency auto 4 kilohertz but the the phone company samples at a kilohertz this is the minimum rate in for optics is the same and for all oscillatory phenomena what happened this is an example here a continuous function can be completely represented by a set of equally space samples if the samples occur at more than twice the frequency of the highest frequency component of the function to capture a function with maximum frequency f sample it a frequency at least two f n is called the nyquist limit in this case blue dot sample on say per wavelengths and on the sample the actual the actual frequency rate we cannot extract information if we only sample one point per oscillation we need at least two to build this similar to what is the original one okay let's see an example here for for the microscope if we have this is the Riley criteria criteria here if we have this two spot we need at least three pixel or two pixels in between if you have more better but at least to see this we need three pixel there in the ccd camera this is what happened when we have the zebra only one pixel you cannot detect some fine details there and this is very important because you can have very good image with your microscope but the ccd camera doesn't match with the with the microscope resolution let's see one example the ccd normally has a 12 12 square a micron pixel using this kind of objective these wavelengths the resolution limit the rillet limit is 0.2 to micrometer this is the resolution that your system is able to give let's see we have magnification that gel is the fraction limit spot with this magnification we have this is a diffraction limit what we have there okay which is the distance of resolution limit on the ccd phase plate so what what do you do okay because it's not possible that this is matching with this we only the the sampling terrain is is not well applied there we need at least to increase this oh to increase this to have more a pixel and to be able to detect this we magnify in this case the magnification is very important we magnify using using a zoom 2x using this this kind of objective an additional objective in your camera and then the resolution limit is now twice the this resolution limit increased twice it means that now we with this we can detect this because it's more than twice the sampling resolution of 12 microns is that clear this is very important point that we have to keep in mind okay but what happened if if you need more resolution than this using your objective and good camera you are applying the nyquist theorem very well but you need more what to do okay as i say shorter wavelength higher index emission liquid in between the the specimen and the objective confocal improve also resolution confocal microscopy using pinhole you can increase the resolution uh we will have some lecture about confocal microscopy professor calling shepherd okay if you can study single spot single molecule or particles in sparse field can find this center at arbitrarily high resolution this is what i say before when we compare different size of the particles you can localize at the center of this by the by the brightness and you can localize this is one of the principles of the localization super resolution microscopy but we can also use multicolor resolution that is not limited by the fraction you have different color it's not limited by the fraction different colors you are able to differentiate between red and blue for example even though that they are below the rally criteria criteria this is what one of the resolution super resolution techniques use use for example for studying uh synastase process in the brain and this is what we will have also in the during the winter college uh and finally we can use super resolution or optical nanoscopic alberto di aspro we will be uh teaching about this topic now i will stop here because i have a second part of this conference uh related with a abbey diffraction experiment or abbey if you say it is as abay is the correct because he's uh from germany okay but before going to the to the second conference uh it's better to have question about this topic the second one will be maybe 20 25 minutes we have time till 12 30 question about this topic please this is very important and this is what is related with the experimental sessions also some questions it's clear i will i will shift to the second conference i can understand for focusing the rule by can understand how to focus or no but there are many rules i don't know what you are referring the uh you have to focus the the obviative in the in the upper part of the of the sample this is a question okay it depends on the on the thickness of the of the sample if your uh your sample is thick maybe you have to do stacks and they and then to collect all the image because as as you saw before the you lose resolution when you uh go in set direction it means if you have very thick samples uh very very thick related with the numerical aperture of the objective uh you have to go in depth taking image moving moving the the the spot focus there and taking image if it's not so takes you can take the image directly it depends and also uh a wish resolution uh unit but there is not a rule there are some tricks there for the person working on this but it depends on the application five minutes to change to shift to the second conference okay we we start with the second part now oh i mistake not this i mistake in the in the name here this is that the abidi fraction experiment is abidi fraction experiment let's see yes yes this is the abidi fraction demonstration not not the super resolution technique in the in the way page also there is a mistake there i have to correct this okay do you know about abidi fraction experiment this is the most important experiment in microscopy hey due to that there is a memorial here in in in jenna and abi was working in in jenna and in this memorial there is this very important equation that we were discussing these days how to derive this equation this is what abi did during that time for demonstration of the resolution equation and the resolution limit and this is what we will do in the lab today due to that i decided to give this conference today okay the minimum distance between two points that still can be uh distinguishable or they can look at different points is related with this very important equation this equation and we have the wavelength you know the shorter wavelength we can go down and also high numerical aperture this is a numerical aperture of the objective okay what is the abidi fraction experiment what abidi for demonstration of this using a white light as miguel said that this equation and and this where did i for white light because they were working with our laser now you have many lasers in your lab scientists but they they work with white light okay what happened when we have the objective length here this is the objective length okay we know that in the back focal plane this is the sample here abit abi locate there a deflation grating with a regular structure because we know that that the that the image is uh is the sum is the result of a lot of points as we demonstrated before we some days and we have the image and if you have regular structure as the deflation grating it's a very good idea for describing the resolution equation this is the the the focus of the image plane what happened exactly in the back focal plane of the objective i say yesterday that the back focal plane normally i didn't bring the objective today but it's this ring that this back we will go in the lab with an objective with very very low numerical aperture in this objective we we are able to to identify this plane because it's very old objective but normally you cannot see there because it's almost inside the objective in the back focal plane but speaking in the in the in words of a Fourier optics this is this is this is the Fourier transfer of this and this is the inverse Fourier transfer okay in the in the back focal plane of the objective what we will have is a diffraction pattern of this grating here and if we use different gratings there we will demonstrate that for example we have there a grating with a 1000 line per millimeters with this grating these are very fine details the the light spread here so much and here also the the first order are good we only will have the central order of diffraction the central order does not any information about the structure of the sample because it's not diffracted and microscopy is diffraction and then interference and we will see in the lab this very clear first with white light with a low numerical aperture objective and with lengths because it's the same but with lengths we can see all this all this plane very clear and we can play there for learning and in this case let's we have we know that the diffraction is related with the wavelength of light and the distance between the the strip of the of the grating we can calculate this if we know this normally we know this the manufacturer say you this distance we know the wavelength we can calculate this angle this is what Abe did he said okay if i if i have and also we will see in the lab using this grating 100 1000 line per millimeter with ana also you have different ratings and we will use a microscope objective that is not able to capture this we'll see no image there but we will reduce this number till we have image because we are able to collect at least the first orders and also we will use different objective and we will see oh with this objective or length we are able to resolve this sample we can construct we can build the image using this length we change with another length oh this length is not able it's due to the resolution and due to that in this case Abe said oh if we adjust that this and this and we have at least the first order oh we have image we will see here the defraction pattern we go ahead there the image will be created here in this in this in this place here we will see the image there intentionally i did this for me with laser because we have laser now and we can see very clear the defraction and interfering i want you to see there defraction and the the spot start to to get together interferes interfere the image very nice experiment and then we can use additional lengths for collecting this we put the length we collect this image and create this image there and the image of the sample will be in different in different in another plane as we studied yesterday we have different plane in the microscope i will have to hear a better length to see this because normally you see the image here and this is not possible to see but even we will see there looking there because we are using laser if not we have to use many lengths but we also will play in the lab with this okay in this case we have we have here the image of the defraction created what happened if we remove this to order when this angle is bigger that the gathering angle of the objective no image formation because we lose the information about the sample the information is in this defraction being and of course in the following orders then no image where is the limit where is the limit what abed abed it for the limit how you you can identify the limit and and this equation oh at the border at the border the two wavelets that i explained before at the border this is the maximal resolution of the system and where is this limit oh this limit is here when this angle here is equal to the defraction angle the gathering angle of the objective is equal to the this defraction angle this is a maximal resolution we can have and doing this we obtain the famous resolution equation very easy the same equation that miguel said and demonstrated and abided many years ago yes of course of course you are limited you you can have very high resolution making a fixed length high curvature but you will have higher aberration and then you need a compromise there surface okay but you can you can increase the resolution and also you can reduce the aberration using some of the techniques i have explained yesterday using correction color for for chromatic aberration using doublets hey in this case i add here a length because we only need now to demonstrate the equation and and to understand about this phenomena but normally this is a set of lengths as i said yesterday 17 lengths in some case more sophisticated objective for correction of the aberration and also you can have very high resolution nowadays because you correct about aberrations okay then this is a the resolution equation this is the resolution limit that is proportional to lambda lambda over the numerical aperture of the of the objective this is the way very simple that abided many years ago for the demonstration of the resolution limit there is a resolution limit depending on the objective and depend on the maximal angle you are able to capture because if this being are good no information about about the structure and we will see this in the lab we will play there with different rating and you will play there with iris because i have a length as i say you in the objective but focal plane is difficult to see there intentionally i add lengths with even very large focal distance and you will look in the back focal plane the diffraction pattern different very nice diffraction pattern circle rings spot we'll have for example there a square by dimensional a diffraction rating that this square line line in this direction line in this direction the diffraction pattern will be similar to what a miguel show the face of the of the of the johnny emel and so on is star as a star of course there is no information in in the frequencies about the face and so on is and you will play there with a slit horizontally and you will block exactly here in the back focal plane you will block this diffraction pattern and then you come and you look at the image and you lose if you block in this direction you lose information in this direction you only will will have lines in one direction and the opposite you block here you have lines only in this direction and we have there are many many ratings for play this is very very i think is the the best experiment that we will have interesting because also you have the possibility to play with this experiment is the same play we have today at the same time this experiment and also image formation with lengths with wide line okay then what i will do is to to divide the the groups five students five students five students i will i will speak about a little about a special interferometry because we will have lecture next week but today we have also experiments there and i need to explain something at least the basics we will have five students with alec Villabona is tutor there i will explain i will explain the theory now and there the experiment you will capture the the video of etanol water evaporating there in 10 seconds you will be discussing with him and in the meantime i will be with other students the five the rest of the five students tutor will be saying up in abidi fracture i also at the first time i will explain for all the students boss experiment then i will leave you with tutors they know very well what to do there and you will play there with different grating with different slit iris because we have also rings the deflation pattern of rings and the image and you can block the center the second and you will see how you lose the image you lose details in the image this is what Miguel did today using mathematics it's a very good combination between theory and practice today theory and practice there in the in the lab and also here with ana consortini defraction and interference this is what is important in microscopy defraction and interference all the images i are created by this but let we finish this this is what again to show you the plane of the microscope but when we have here we will have this great in there one one thousand lines per millimeter we we are not able to resolve this there is any length you will see that the defraction pattern is very very the second the the first order are far away from from the central order it's not possible we need high numerical aperture maybe 1.4 i don't know oil immersion i didn't check but i don't have but the most important thing is is the the theory when you when you know this you can do whatever you want you can build a microscope as i did there okay then we have a condenser here condenser iris as i explained before we have the condenser length we have a parallel beam of light this is very important a parallel beam of light through the microscope to have defraction pattern here the defraction pattern here and of course the image far away in some case in this case we need a second length a tube length to create the image and maybe we can see directly at the defraction pattern in some case in some case we need an additional length doesn't matter we will see they are together with the defraction pattern the image of the of the source because they are simultaneously in plane they belong to the fill fill planes of the microscope not to the image plane there are four planes image plane four image plane okay this is what speaking in in words of a Fourier optics this is what happened there with the this defraction pattern that you have here a this is okay what happened here okay it's an example that what what we will see there spot the fraction fraction there image formation okay this is what we will block we lose information about the image okay and this is a again some better illustration of the of the experiment in which we show the planes and the orders this is the the orders of the defraction here one minus one and one and this is what we will block there here a better illustration what happened after the fraction after the fraction we have interference and the image is created in the intermediate image plane okay then a besay set the microscope image is the interfering effect of defraction phenomena very clear for this conference it's all i will give you the opportunity to give question and then we will speak about the experiment the rest of the experiment there question please or very clear or or not clear i think it's very clear and after the michael conference of course and after the experimental session more clear i think my main my idea is everyone not only to use a microscope to build a microscope you can if you learn this and you play there in the lab i i am sure and this is my objective you to build a microscope or to modify a microscope not to buy only sometimes the objective you have to buy but even you can use after that you have a complete set of a lecture you will have lectures here with michaela to scanning Javier Ramirez and Jorge Alec Villabona how to motorize a microscope to define or to focus the sample in the correct place to capture the image to move the sample capture image to do a map of fluorescence or nanodiamond samples of course we don't have here a fluorescent microscope but doesn't matter the most important is the theory and you know now the theory about the the fluorescent also and to to to add this element and this this theory or this practice to the microscope is easier if you if you domain this this concept and this he will teach you about the software the hardware detectors motors and so on okay question okay now let we have 20 minutes i will speak about a speckle we'll have also thermal length three sparing per day we have another tutor there is sulima i was explaining that five students taking a video because we are not able to have all the students at the same time there it's impossible only five and you have the possibility to play there because some of the students will be waiting there doing nothing and i don't like this i like all of you to be busy and then the the course was designed in this way that five students working with uh is is not so easy but with it alex recording videos about speckle that i will explain now then five students working with me in a video fraction playing their slang and image formation there are two experiments at the same time there but are similar then the five students here come with ali to record video and these students come with me suliman will be not busy during during that time he will be after that five students go there with suliman thermal length i will not play thermal length because we'll be many later next week then they working with suliman i will explain also thermal length there before and five students processing with ali in different rooms processing the speckle video information with a program in mad lab that we develop and we process the evaporation of ethanol water because ethanol water simulate a parasite medium by a biological medium is similar when you add a drug to a parasite to kill them they get stimulated for example tripanosomacluse they get stimulated and the activity increase and you have a high activity then the activity start to decrease due to the action of the drug and then when no activity is a constant and in parasite we have obtained this in the paper we publish and we don't need parasite you only need the procedure you don't need only to use the setup how to use how to process data and then if you if you want to use parasite bacteria i don't know you can use the idea is the program in mad lab divide separate the the video in frames in frames pixel by pixel then we apply algorithm there are many algorithms in speckle interferometry you apply the difference between two images pixel by pixel give a number second third image give another number and so on of course if you have intensity pattern here moving and in the second moving in different the rest is a high number but when the parasites are dying or the tunnel is almost evaporate no activity this pixel has the same intensity intensity that's the the next one and so on when you subtract what happened zero intensity and this is proportional to the activity versus time but i didn't say what is speckle interference when you have a laser let's see when you look at the wider spot there in a in a surface that is not speculates a regular surface you have a reflection in multiple direction and this multiple beam scattered beam interferes and gives you a pattern of multiple points brighter and dark points if the surface is static this pattern will be static if the surface is moving this pattern will be moving proportional in proportion to the to the movement of the of the medium biological medium then you can account for this you can account for the for the movement for the mobility of the of the medium processing the speckle pattern and give you very very useful information it's very uh with low cost sparing you need of course you need a coherent laser it's better to use hill union laser because the coherent length is better but we also use a less coherent laser if you don't need a precise measurements but it's better to use hill union laser because the coherent is maximum there and you have then a length you you must illuminate or the sample but in this case we don't use length we use a diffuser because the diffuser did multiple scattering and then because you need also even illumination onto the sample because if you have speckle image there intensity and you process and the intensity is not the same you have another variable there the intensity of the pixel and this is not good and we did with diffuser very even illumination there we don't need a divergent length after that maybe attenuators some attenuators not to saturate the the camera and a ccd camera there collecting the image very very simple setup but with this setup we publish in in december a paper in in plus one neglected tropical disease in in tripanosoma cruci parasites this setup costs less than one thousand dollars maybe a ccd camera but you can use also webcam even we did it with webcam maybe when you you you pull is the paper some referee will say no it's not professional the results are good the results are good but he will say no and read the paper because it's a webcam what to do but you can have very very good results low cost speckle image as also we will do is a heavier very low cost components you can have very good results you can do up to automatization of as you did there of a fluorescent microscopy for fluorescent you only need the the objective to excite the sample at specific wavelength to collect with the the correct resolution the fluorescence to detect in ccd camera and the question is how to move the stage to put the sample in the in the maximal focusing to have a good image and if you need to collect a stack of image in depth to move the focal length or the sample and to collect this image also using this software that you will teach and have a map of fluorescence as we show in the in the first lecture about the cell with the nucleus in red the actin filaments in red the chromosomes in green adding there some stain as in brana dish or we'll teach about green fluorescent proteins and this is the idea i think we have still some minutes for questions or comments it's clear what we will do there in this two experiments speckle abidi fraction image formation maybe we can comment about thermal length five minutes a thermal length we have a setup there also is a technique that normally is a spectroscopy technique in which you excite the sample with a laser and of course the sample moves absorbed at this wavelength but normally it's for samples that does not present fluorescence they are mainly they relax this energy mainly by thermal channels and if you have a microscopy you cannot detect this kind of this energy they are create a refractive index grading due to the temperature grading because you have they are if you have a liquid you have a different temperature where the temperature is higher because being Gaussian focus you have there the liquid span less intensity far away from the focus you will have a less temperature higher density this is the length divergent length but this created temporary because you chop there there is excitation no excitation and then with a second beam red in this case path there and where the beam pass there no thermal length no excitation the beam is parallel when you have the effect the beam spread diverge and you put a detector there and when detector at the detector reach this kind of signal of course parallel beam higher intensity diverge you lose intensity in the detector for that we have a pinhole there as in confocal microscopy and then you can detect this variation and you have a signal you have a signal that is proportional to the properties of this medium is pro is proportional to the absorption of the medium to the concentration to the thermal properties and so on you can characterize this medium if you convert if you convert this thermal image in colors you are you are doing similar to fluorescence is similar to fluorescence microscopy instead of taking fluorescence you are taking thermal length for example you have a cell you have a cell from the mitochondria i don't know you have a different thermal length because the mitochondria has different components absorb different from the all mitochondria you have similar signal and for other parts you have another signal because the absorption is different if you build this in colors you have the image of the cell it's the same but with very high resolution because this technique is with very high resolution because we have a resonator and then we increase the the the sensitivity no resolution because in this case this is also the sensitivity you can have high sensitivity that you can detect the signal but the resolution is not so high the resolution depends on the objective you are using there and we will show experiment there in a semiconductor sample one micron thickness this sample was built by a electron microscopy and they reported that it's one micron but really it's not one micron it's 99, 99, 99, 500 microns maybe in one part and less than this in one part how to check that it's difficult but this system is able to to differentiate but for that you need high resolution to do a map and you will see in the lab moving very precise five microns five microns the signal is different because the signal is proportional to the thickness then we are able to detect nanometric difference in a very very thin sample when we have an homogeneous sample we don't need resolution because we only need to excite the sample and to get the signal of this average and to have a value of absorption in average volume we don't need resolution this is the difference between normal thermal length and thermal length microscopy the same applies for all other systems you need a resolution when you have there fine data that has some difference in absorption in composition there if not quite to to have high resolution you don't need and we will see this in the lab question please no question everything is clear a comment can allow us to adjust the resolution according to our needs and it's not a technique just used by a high resolution acquisition but it's also used in local for example when you use to see that I am using a photocell or photo dial to the decline and you can actually acquire images point by point by using the mechanical mirror this is similar this is localization microscopy this is a super resolution technique you are doing localization microscopy okay that's all thank you very much for your attention see you at 150 five minutes before the the boss is not waiting for nobody he lost the boss last time the driver will left then here they have a strict timetable