 I think I have to attach power with my laptop. I also learned it now, don't worry. We are the same because it's not my field. Thank you, Miguel. So where is the power? I saw you. Thank you. No, that one. Okay. Now you represent me. The next session is Professor Ibrana Ashraf. She's going to talk about the glory. That's nice and short. Yeah. Because if you don't know the field by yourself, don't mess around with it. Thank you, Miguel. Thanks a lot. So time is, so it's about fluorescence and we will start with the fluorescence and by the end of this lecture, I'll try to explain to you green fluorescence protein, which they asked me to do. But my idea was to do the cellular biology first instead of doing the green proteins because what is a protein? I don't know how many of you are from biology, but I'm not from biology. For me, if somebody was giving this green fluorescent protein by protein, what is protein? These are the common question which I could have come to my mind. So, but anyways, let's start with the fluorescence, what is a fluorescence? And then I will explain some kind of molecules. Those have these kind of properties which fluorescence, do fluorescence. So just basic of fluorescence, what is the quantum yield of fluorescence, fluorescence lifetime and fluorescence in nature. Applications of fluorescence and then I will end at the green fluorescence protein. Okay, so fluorescence is the emission of light by a substance that has been excited or that absorbed light for any other electromagnetic radiation. So basically a substance or a molecule or an atom that absorbs light or any other electromagnetic radiation, but it emit light. That it emits in the visible part of electromagnetic spectrum. So the emitted light has a longer wavelength. Longer wavelengths mean shorter, the less will be the energy. So it has a longer wavelength, it will absorb in a higher wavelength and then it remit in a shorter wavelength, longer wavelength. So therefore the lower energy then the absorbed radiation, I am sorry I am confusing between frequency and wavelength. So the most striking example of fluorescence occur when the absorbed radiation is in ultraviolet region of spectrum and the emission is in the visible region. So we also know these fluorescent light which they usually have a first force coating inside. So they absorb elective UV lights and they emit visible part of spectrum. And sometime maybe you have heard that in a graveyard people can see light, it was glowing light at night or something. So we all have phosphorous in our bones. So when the decay process starts and if the grave is opened by some mean or some animal has done that. So you can see a glowing light and if you are a superstitious there are many other stories related with these kind of thing that oh there was a glow in his or her grave or these kind of things. So fluorescence material sees to glow immediately when the radiation source stopped. As soon as we are making an excitation from a ground state to excited state with some kind of visible light or some kind of another electromagnetic radiation. So it as soon as it absorbs I will explain to you what happened and then it emits light but if we stop excitation or stop the source which give energy for this substance atom or molecule to get excited from its ground state to excited state it sees after that it will not glow. So it is a very short time phenomenon which fluorescence and then we will see what is the phosphorescence and other things. These are the fluorescence glow from that can be a chemical dye or fluorescent dye which can be mixed into this solvent and when it is illuminated they are emitting these colors. They are different dyes that would mean G6 and which people use for dye lasers and here we will encounter with the term which is called fluorophores. These are the molecules that absorb light or again the electromagnetic radiation and emit light. So different fluorophore or different types of these molecules which has this property of emitting light after absorbing electromagnetic radiation or visible part of electro-spectrum they are called fluorophores and they have each fluorophore has a specific spectrum emission and absorption first it absorbs and the absorption spectrum is also called excitation spectrum. So there is a wavelength which one specific fluorophore absorbs some radiation and then it remits and as we know in the fluorescence that the thing that they emit is longer in wavelength. It absorbs higher energy light but it emits in a lower energy light. Maybe if it absorbs blue light it will emit in red. We will see in a minute what happens. So different fluorophore absorbs different wavelength of light. Each fluorophore has specific excitation which is the absorption spectrum and it also has a specific emission spectrum. So it depends upon that kind of fluorophore which we are using whether it is this dye, chemical dye or it is the some cell or some protein which absorbs light and remits light and they also they have a specific excitation and absorption spectrum. So fluorescence occur when and we know that there are two kind of if we are talking about molecules. We know there are singlet states and triplet states. So what is a singlet state? These are electronic transitions. S naught is the ground state. Singlet state is when the spin are in those orbits according to the poly excretion principle. One has spin up the other one is spin down. So spin as a total is equal to 0. So multiplicity will be 1. So this state is called the singlet state. And the triplet states are we encounter when we are dealing with molecules like yesterday I told you molecule experience different kind of vibrational rotational electronic transitions and they are under pressure under four different kind of forces. And during that process sometime there are state in which electrons are both are with spin up or both are in spin down. If they are spin up it gives you a spin of half plus half is 1. So when 2s plus 1 you do it you will get a triplet state and those triplet states are also called forbidden states because they are not following the proper rule. So it is for when an orbital electron or molecule or atom relaxes to its ground state by emitting a photon from an excited singlet state. Here you can see this is the absorption or excitation. There is an S naught plus you shine this electron from with the photon which lies in its absorption spectra. Then there is a finite probability the electron which is in the ground level which get excited. This electron should be having this frequency h nu should be in its absorption spectra. So then it will get excited and the S 1 is the first excited state and upon emission this S 1 will move the electron will move down to its ground state. It will emit a photon of longer wavelength because some of this energy is decayed like a non-radiated wave. It came out of as the heat or it gave some kinetic energy to the neighboring molecules. So this electron photon is not equal to this photon. If they were equal then that process we call as resonance fluorescence the absorbed photon and emitted photons are same. So here in fluorescence if it absorbs the high energy photon it will release a low energy photon and rest of the energy will go as the heat or other things. So where h is the Planck's constant S naught is the ground state of the fluorescent molecule these are electronic state. S 1 is the first excited state. The specific frequency of excitation and emitted light are dependent on the particular system if it is a fluorophore of one kind let us say A kind B or C so it will have its own specific emission and absorption spectrum. So molecule in S 1 can relax by various different pathways. It can non-radiated we know that what is the non-radiated transition. Yesterday we saw what is a spontaneous emission. Spontaneous emission is radiative because we get a photon which is not in phase and all this spontaneous emission not necessarily is in phase but we know that the stimulated emitted photons are in phase. So if an electron is in excited state its energy it gives due to collion electron lost its energy or due to some another matter it lost its energy then it will come down to the stable ground state without emitting a photon. So that kind of transitions are called non-radiative transitions because there is no radiation that is involved. So in this relaxation the excitation energy is dissipated at heat or the vibration to the solvent if there are dyes like I show you this cartoon where you see that these are the dyes which are mixed in a kind of solvent they can give heat to those solvents or to the neighboring molecules. So in that case there is no photon I will explain that again. So phosphorescence what is the difference between fluorescence and phosphorescence? Anybody sorry triplet state. So even you stop the source which is giving the light you can see the phosphorescence. So excited organic molecule can also relax via conversion to a triplet state which may subsequently relax via phosphorescence or by a secondary non-radiative this thing. Let me go to this cartoon I will explain this. So this is the ground state this is a Blonsky diagram and the people who are doing atomic physics I am sure they know about it. So this is the ground state this is the electronic ground state and the ground state is represented by this is singlet state by S naught. So electron is here. So if you incident or shine this molecule chloropore with the kind of photon of energy which lies in its absorption or excitation spectrum it will absorb and end up either in the first excited state or in the second excited state as to is the second excited state. So these are the electronic levels and these others are the vibrational and rotational level in case of a molecule and chloropore is a molecule. So what happens? If these states are unstable as soon as the electron landed at this point or this point it vibrates down to the nearest possible electronic state which is stable as compared to these vibrational and rotational levels. So we all know that nature prefers to state the lowest possible energy. So this was the lowest energy we probe or we provided external energy to rise this photon from this point to this and here in these vibrational levels they will rotate or vibrate and get down to nearest electronic transition. From there they have two options to read decay non-radiative way in which there will be no emission of photons or radiative decay. So this radiative decay which is from this level is called a fluorescence. The decay from this electronic level to either of these vibrational level this red line shows the radiative decay or they can decay non-radiative by giving energy to their solvent by giving energy to their debers and in this case there will be no photon emitted. Another method is this point in which the electron loses energy during rotation and vibration is called internal conversion and it follows a rule that is called Kassasha rule I think there is a point and according to that rule they are immediately as soon as the electron land in this position or this position it immediately rotates and vibrate and get down to nearest electronic level. So this is called internal conversion and if electron is here it can also make transition to triplet state and this process is called intersystem crossing. So electron in one of these states can make a transition from this singlet state to this forbidden state which has opposite parity and it is triplet state so it can come here. So if electron is here then again it has two parts to follow either it will decay non-radiative way to this ground state or it decay in a radiative fashion and this radiative fashion is called phosphorescence because it is and you can see the time scale electron get excited and absorption take place into raise to power minus 15 seconds because it is the transition frequency and it is near optical region or visible region. So its internal conversion is again very quick it is in between 10 raise to power minus 14 to 10 raise to power minus 11 seconds and this phosphorescence is of the order of 10 raise to power minus 3 to 10 raise to power 2 seconds which is very long as compared to this fluorescence. So phosphorescence even if this excitation or this source is stopped you can see the phosphorescence but for phosphorescence electron has to be in this forbidden state then it will do the phosphorescence and then this electron can also go back to this singlet state and again via these are two directions inter system crossing is this way and that way if electron is get back to this singlet state then it can again it have two paths non-radiative decay and radiative decay in this case because you excited this electron earlier and now you will get a kind of fluorescence that is called if it selected a path to go in a radiative way you will get a photon and it will have the energy difference between these two levels and that will be a bit delayed and this is called delayed fluorescence if this from inter crossing it get back here or if it will decay non-radiatively then it is the same radiation that transition so this is the difference between fluorescence delayed fluorescence and phosphorescence phosphorescence is always from triplet state or forbidden states ok so this was the phosphorescence and this is fluorescence quenching this means the relaxation from S1 can also occur through interaction with the second molecule so quenching is basically a process which decrease the fluorescence intensity so it can be collienic quenching it can be fluorescence quenching in that case because the molecule A has given its energy to another molecule B ok so then as I mentioned because these fluorophore absorbed in between their excitation spectrum or emit in their emission spectrum so if there is a kind of fluorophore whose absorption is represented by this so it has a maxima over here it means that any photon having this wavelength because here this is wavelength corresponding to this if there is a photon having this wavelength it is has a most probable kind of way to that it will absorb that photon and make a transition from ground state to the first excited state but in between all these wavelength of this fluorophore can be excited this is the most probable and when it emits as it makes a longer frequency a longer wavelength so we know that if it is it absorbs in blue and it emits in red so red has a longer wavelength as compared to blue and red is low in energy as compared to blue so this is its emission spectrum and this is its absorption spectrum the difference between the peak of yeah sorry intensity of absorption yeah are supposed to be the same no no no because it has it is not a single line it is not a single line if it is a single line and if emission and absorption is the same then it is called resonance fluorescence in fluorescence it absorbs higher energy and emits a lower energy invisible spectrum so this these peaks are actually the point or the wavelength which is most if you apply shine that fluorophore with a photon having a wavelength in this oh sorry in this region so it will excite the fluorophore more easily and when it de-excites it can emit any photon between this red curve so the difference between peak of the blue and this is called stroke shift and this is also called red shift because it is shifted towards that so resonance fluorescence if the emitted radiation has the same wavelength as the absorbed one so if the photon if it is a red light photon it has been absorbed and then it emits again the red photon then it means there is no stroke shift so they are in resonance the absorption of photon and emission of the same kind yeah yes yes yes yeah yeah yeah because this is like this like the question you are asking is this is absorption after that it rotate or vibrate down to this excited state so it lost some energy as heat or some other form but from here it will emit so this internal conversion is actually the time electron state in excited state is basically it is life time and that is very short in that case if it makes a transition to this triplet state and then when it will emit it will be a delayed process but one should know that it is fast fluorescence or delayed fluorescence intra band no no it is not possible yeah because these are unstable it is not possible usually the fluorescence the energy difference between two electronic levels is lies in yesterday we saw that it lies in the visible range so that is why we need for fluorescence is something which we see it is in the visible emission is in the visible part no energy is because when it make a transition from this first electronic excited state to it will emit a photon which will be yeah that is the fluorescence so the other one is not fluorescence because that is the energy it should be in the visible part of electromagnetics you have some question okay so now what is the quantum yield of I think I have done with this slide okay so quantum yield gives the efficiency of a fluorescence process it is basically the ratio of number of photons emitted with number of photon absorbed so if we know that number of photons efficiency will be 1 or 100% if the number of emitted photon is equal to the number of photons that are absorbed it is not possible so each photon absorbed result in a photon emitted then it will be the maximum fluorescence quantum yield compound with the quantum yield of even 10% are still considered quite fluorescent it is not that they should have 100% fluorescence efficiency or the quantum yield even the 10% are considered they are fluorescent so there are many process that affect the quantum yield these are the same kind of process which affect this quantum yield you can have dynamic collingial quenching if there are molecules in a solvent or in the gas form they collide with each other so there is a possibility if it is going to transition the energy can be transferred to the next molecule it can be resonance energy transfer a mechanism describing the energy transfer between two light sensitive molecule that I think I do not know I was not present in Barthos whether he explain this fret or microscopy or film or these kind of thing I do not know so he will do I think so internal conversion as we see that internal conversion can affect the efficiency quantum yield and inter system crossing if electron from singlet state moves to the triplet state it affects the its efficiency and the quantum yield or it can go back to from the triplet state to the singlet state and in that case we will have this either at the non-radiated transition or are delayed fluorescence ok so the another parameter related with this fluorescence is its life time so what will be the life time if this is the ground state that is s naught and it absorbs the photon in between its excitation spectrum and it make a transition from this to any vibrational level or rotational level up there so now it try it vibrates down or rotate down to this closest possible electronic level so the lifetime of electron in the excited state is actually its life time so as it moves in due to this internal conversion it moves very quickly from these unstable states to reach this electronic level so that time is very short so the fluorescence have refers to the every time that molecules because if the electron of a molecule is in excited state it means that molecule is in excited state or in excited state actually it corresponds to that atom is in excited state so in the excited state before emitting a photon photon it will emit or do the non-radiated transition only when it is from in its first excited or second excited electronic state so fluorescence typically follow the yeah yeah ok let us go back here so this is electron is excited from here to there or from here to here so it is excited now the lifetime as soon as the electron land there its lifetime starts so due to internal conversion it vibrates down to this so the time it span here it is its lifetime yes it is even the internal conversion is you can see this internal conversion is of the order of there is to power of minus 14 to fluorescence is of the order of nanosciences yeah sorry lifetime depends upon that fluorophore or on the excitation because we can excite this thing between this any we can send any photon of having wavelength between this peak this distribution so it depends on that also the electron can get land here here or any above so it will take if it is landed here so it is lifetime to get here will be bigger as compared to the this one so if electron so I was telling him that if electron is here its lifetime will be shorter as compared to the electron which is here because there is s2 or it will take a little more time to get down so so this fluorescence follow the first order kinetics what is the first order or 0th order what will be the 0th order kind of equation this we all are familiar with this thing how did you active yes so what will be the 0th order this is first order I said that it is first order sorry so what kind of curve I will get so that I can understand it in this case it will have an exponential decay we have an exponential factor over date that depends upon the decay constant and the time so in case of 0th order we will have a linear behavior so here we will have this this is actually this s1 stand 0 for is at the initial concentration at time t is equal to 0 s1 is at a later time t and it gamma is the decay rate or and that is equal to the inverse of fluorescence lifetime so if I see the fluorescence lifetime various radiative and non-radiative process can depopulate the excited state it can be radiative it can be non-radiative it can be inter-system with a triplet state so it is a sum of all these things so if we consider there is no inter-system crossing so we can have a total decay radiative and non-radiative so if the rate of spontaneous any of other rates are fast the lifetime is short as soon as the atom comes to down lifetime will be quite short so for commonly used fluorescent compound typical excited state decay time for photon emission with energies from UV to near infrared are within the range of 0.5 to 20 nanosecond it is in the lifetime is in the range of 10 raise to power minus 9 so the fluorescence lifetime is an important parameter for practical application of fluorescence such as the fluorescence resonance energy transfer this is the FRET and fluorescence lifetime imaging microscopy Ambarthu was explaining something yesterday it is the the economics film and for that is FRET so I think he will explain or you will listen these things during your winter college this is again the same thing this is non-radiative transition between these vibrational or rotational level this is the absorption this is fluorescent and the system relaxes vibrationally eventually processes at a longer wavelength because the energy is less some of the energy has lost as a heat or during non-radiated transitions so the emitted photon will have longer wavelength as compared to the absorbed one so there are several general rules that deal with the fluorescence this is CASASHA well it would it according to this rule details that quantum yield of fluorescence is independent of the wavelength of excitation radiation why because if you go and see that it does not matter that it has been excited from here to here or here to ever it eventually land to this first excited state nearest electronic state so it does not matter that electron or it does not depend on the absorption spectrum or something so this rules does not always apply and it is violated many simple molecules even so the fluorescence spectrum show very little dependence on the wavelength of excitation or the absorbing radiations so then there is a mirror image rule that for many fluorophores the molecules which emit light the absorption specter is a mirror image of the emission spectrum so there are some fluorophores which have this kind of behavior so there are many natural compounds that exhibit fluorescence and have a number of applications so how many of you are from biophysics so biophysics okay so you know much better than me how to use these bioluminescence and biofluorescence or sorry okay there are many natural compounds that exhibit fluorescence and they have a number of applications so one of those phenomena is biofluorescence then we have biofluorescence, biotic biofluorescence biotic fluorescence what is a biofluorescence have you seen fireflies biological system the fireflies is an example of because you see the light yeah plant them they call it okay so is the absorption of electromagnetic wavelengths from the visible light spectrum biofluorescent protein that is why I am telling you that all these biological system depends upon the proteins which have certain colors similarly the green fluorescence protein so I wanted to see that what is a protein actually in a living organism and the re-emission of the light at a lower energy level they follow the same even if they are biological system or even if they are cell or any organism they follow the same rule as fluorescence in other kind of substances which have this property this cause the light that is re-emitted to be different color then the light that is absorbed because we know that it absorb higher energy and emit but in between your visible range then you are able to see those fluorescence okay stimulating light excite an electron even if it is a biological system or an organism it is electrons are excited raising energy to an unstable level unstable level is the same like we have those vibrational and rotational levels and then it will fall down to the first stable level and from there it will emit light this instability is unfavorable so the energized electron is returned to a stable state which can be like we see we saw in this cartoon that it can be this electronic transition where after these non-radiative transition the excited electron will land here and from here it can make radiative non-radiative both kind of transitions and the same rule is applied in biological systems or and natural fluorophores okay this is this return to the stability correspond with the release of the in the form of fluorescent light these emission of light is only observable when the stimulant in light is still providing light to the organism because in fluorescence if we want to see the fluorescence you need your source to be on you can see phosphorescence in the absence of that source but for fluorescence if the excitation source is on so chromatophores are pigment containing and light reflecting cell or group of cells fluorescent pigment cells that exhibit fluorescence and function as thematically are similar to regular chromatophores and fluorescence can be found in the skin in the jelly face or just below the epidermis amongst the other so what was the where we they get the first like Ambartho mentioned about green fluorescence protein yesterday and he mentioned the Nobel prize people got for that green fluorescence protein and you remember what was that so about did they get the first green fluorescence protein it was a kind of jelly fish so they killed I think I was when I was preparing that I saw the on internet that they killed millions of millions of jelly fish there was a Japanese scientist who was they were and they remove a kind of ring and then they saw that it has these kind of proteins now they inject that protein can be used as a marker for to that it's a fluorophore and there are two kinds of fluorophores in nature you can say that it's exo or endogenous exogenous is you can inject some dye that can do the fluorescence and endogenous is that inside your there are some chromatic force which have this tendency to do fluorescence like the green fluorescence protein which is present in a kind of jelly fish okay and then we have a aquatic bio fluorescence what happens in under the sea these jelly fish of course they live in sea or in water or in lakes so water absorb light of long wave length we see that why it absorb light of long wave length how we if you go there and see this adrodic sea so you can find that it looks blue so it it absorbs red and reflects blue so water absorbs light of long wave length means longer than blue so less light from these wave length reflect back to reach the eye so therefore warm colors like red orange from the visual light spectrum this is again the visible part of electromagnetic spectrum appear less vibrant at increasing depth as you are going inside the water the intensity of the light decreases and it decreases around 10 fold with every 75 meter of depth so but it absorbs longer wave length so there are certain wave length which are not absorbed by water so you can see this bio fluorescence inside the sea rivers and lakes so water scattered light shorter wave length so shorter wave length is blue or green so meaning cooler colors dominate the visual field in phoetic zone phoetic zone is the kind of zone in sea or water where almost all the photosynthesis take place and the 90% of all marine life live in this zone so they have those kind of light and the photosynthesis is taking place so there is a great balance between oxygen and carbon dioxide in this zone so it scatters the light of shorter wave length that why we see that it is blue or green light intensity decreases 10 fold with every 75 meter of depth so at the depths of 75 meter below sea level light is 10% as intense as it is on the surface it is only 1% as intense as 150 meter as it is on the surface so as you are going down and down the intensity falls off as any type of fluorescence depends on the presence of external source of light that you see that you have to give energy or you have to excite the electron from down level to excited level so you need external source of light so biological functional fluorescence is found in the dephiotic zone where there is not only it is the zone where as I mentioned earlier it is the zone where there is a good balance between oxygen and the carbon dioxide and also there is a photosynthesis is taking place and 90% of marine life in that zone so there you still have a kind of there is only enough light to cause biofluorescence and but enough for other organisms to detect it so if there is a kind of jellyfish it is in that zone of sea you can it fluorescence and you the other animals or other living things which are in that region can see that phenomena occurring that fluorescence can be seen so the visual field in the dephiotic zone is naturally blue so colors of fluorescence can be detected as bright reds oranges yellow and greens so these are the color you can see from that aquatic biofluorescence so gemstones this is then there are the other things which are which occurs in nature we all know about the gems we all know about this highlighter you use at home or you use in your classes to highlight your book or your notes those are also contain these kind of fluorescence things which you can see so gemstones minerals may have a distinctive fluorescence or may fluorescence differently under different short wave ultraviolet long wave ultraviolet visible light or X ray so gemstones have different colors or different kind of fluorescence that you can detect that if you are using short wave ultraviolet maybe you can see different phenomena and if you are using a visible light or X ray you can see different colors so crude oil that is the petroleum fluorescence in a range of colors from dull brown for heavy oils and tars through to bright yellowish and bluish white for very light oils and condensate so organic solution such as I am really bad in this biological I never like to study biology because of these different difficult names and seen or still being dissolved in benzene or toline and fluorescence with ultraviolet or gamma rays in radiation so fluorescence is absorbed in the atmosphere when the air is under energetic electron bombardment so there is another phenomena that occurs in nature and we can see on our north pole and south pole what is that there is a phenomena yeah so there is a north due to the solar wind and the interaction between solar wind and the magnetic field of earth and you see these auroras I should have put one okay these are the gym collections in cases such as the natural aurora the high altitude like nuclear explosions and rocket born electron gun experiments the molecules and iron formed have a fluorescent response to the light so the vitamin B12 fluorescence because these are the things why it is important if these have medical applications if you have got forbid the deficiency of B2 you see the spectrum and you can analyze that it is missing or B12 is missing or these kind of different deficiencies you can detect by the spectroscopy so tonic water blue due to the presence of Q9 highlighter ink is often fluorescent due to the presence of Pyrene and bank nodes postage stamps credit card often have fluorescent security features you have seen there are usually now when it was this holography is also used for few things but I think they have these kind of fluorescence images for these they are real or somebody is doing the poetry so lighting you can see that the common fluorescent lamp relies on fluorescence contain a coating of fluorescent material that is called phosphor and there is also another application which is more common than all the others which is the optical wideness for laundry detergents actually emits blue light when irradiated by the sun it looks the white to appear more white you are right absolutely right because then you they have used that what is the name for that the name of the compounds don't remember their name but I have used them to build ultra luminescent dilacers by exciting them with nitrogen plasma which emits on the ultraviolet region that's good so you called the phosphor which absorbed the ultraviolet and reabids visible light so fluorescent light is more energy efficient than these the other lighting elements of milky color and these spectroscopy it's usually I think the microscopy and the spectroscopy you need usually the setup of a fluorescence as they involve a light source we need a light source we need a monochrome matter to and then we need a sample then we need another filter so it's basically which emit many different wavelengths of light a single wavelength is required for proper analysis so you know that you are using this sample cell or this is a fluorophore which has this absorption spectrum so and then the chosen wavelength is passed through the sample cell so light is passed through excitation monochrome matter you pass through and then it is more focused for that sample that it is mono means single and chromatic means frequency and that the chosen wavelength is passed through the sample cell after absorption and reemission of the energy many wavelengths may emerge due to the stop shift because we know that the emission and absorptions are those band and they have they have a difference of redshift so to separate and analyze them the fluorescent radiation is passed through an emission monochrome matter and observed selectively by the detector so this is the kind of how you do the analysis by using spectroscopy the biochemistry and medicine fluorescence is the life sciences you generally as a non-destructive way of tracking or analysis of biological molecules by means of fluorescent emission at a specific frequency where there is no background from the excitation light as relatively few cellular components are naturally fluorescent that is why this green fluorescent protein is a good marker they inject and I will show you in few slides that now they can even producing animals which have this fluorescent effect so a protein or other component can be labeled with an extrinsic fluorophore it means that it is exogenous fluorophore that you can I was reading that because you know that if there is an antigen antigen is an external toxic substance or it can be a this bacteria virus and then in response to that antigen our body produce or our plasma cells produce antibodies so if you join that antibody with the fluorophore that can be a dye or something or a green fluorescent protein then it can be it can act as a marker and you can label your target cell and many other things and these antibodies are in the shape of they are called by molecule or something like that so sorry labeled with an extrinsic fluorophore it can be a dye or it can be a quantum dot or a small molecule to use as a marker so when scanning the fluorescent intensity across a plane one has a fluorescent microscopy of the tissues cell or a sub-cellular structure so I needed sub-cellular structure to know we will do that in today's time this my cellular biology so that can be done by labeling an antibody as I mentioned that you label an antibody with a fluorophore with a fluorophore and allowing the antibody to find its target antigen within the sample so antigen is like I had told you it is an invader and antibody is the response of our immune system react with against that antigen so they attach to each other if you attach the fluorophore with your antibodies you can target that antigen because it will attach with that part of yourself so labeling multiple antibodies with different fluorophores allow visualizing the utilization of multiple target within a single image so you can use this microscopy which is you are using some marker fluorophores with your antibodies and that can be very helpful to detect or see things in the living organisms so film like I mentioned earlier is the Florence since lifetime imaging microscopy can be used to detect certain biomolecular interaction that manifest themselves by influencing fluorescence lifetime so these are the pictures of course I got from the web and detection of co-localization using fluorescence labeled antibodies for selective detection of the antigen of interest using specialized software such as there is a kind of software which can be used to detect or visualize these fluorescence labeled antibodies so okay this is picture my student put in that so now we will move to the green fluorescence protein and I am sorry I don't know the name of those amino acids and anything so the green fluorescent protein GFP is the protein composed of 30 amino acids residues that exhibits bright green fluorescence when exposed to the light in the blue to ultraviolet range because you know that if you are doing imaging or anything with visible light range it is always safe it is non-invasive because ultraviolet can harm your skin or other things but visible light is safe and this imaging techniques by using bright GFP because you have to expose to light from blue to ultraviolet range so ultraviolet is at the boundary mostly so it is more or less safe although many others marine organisms have similar green fluorescent protein GFP traditional refers to the protein first isolated from jellyfish aquaria Victoria so the GFP from aquaria Victoria has a major excitation peak at wavelength of this thing and minor one at so it has broad spectrum so it has a wavelength peak at this major peak and minor peak so it emission peak is longer wavelength as we see that fluorescence the emission is at longer wavelength which is in the lower green portion of the visible spectrum so when it will emit you can see the green light so the fluorescence quantum yield of GFP is 0.97 which is quite good it cannot be this is aquaria Victoria poor jellyfish so this was a kind of ring first they were doing it with a kind of knife I was reading and then they make special scissors to the attack this circular ring from this jellyfish this makes an excellent tool in many forms of biology due to its ability to form internal chromophore is again a kind of part of the molecule that give you a distinct color so in cell and molecular biology the GFP gene is frequently used as a reporter of expression it has been used in modified form to make biosensor sensors are something which you can track things it can be the changes in your DNA it can be changes in your genes chromosomes and these this in modified form this GFP can be used as a biosensor many animals have been created that express GFP so they have extracted this green fluorescent protein from these jellyfish and then they have added to different animals and introduced in animals and other species through transgenic techniques this is the genetic engineering by using genetic engineering they have injected this green fluorescent protein to other animals and maintained in the genome genome I will explain to you what is genome in my last lecture and that of their offspring that even if when they reproduce they also have this green proteins in their genes so this is the name of the scientist who got this Nobel Prize in 2008 for discovery and development of green fluorescence proteins so they also this is a green fluorescence protein and it has a complicated shape is composed of again amino acids each monomer composed of central alpha helix surrounded by 11 standard slenders of anti-parallel beta sheets so it is a quite complicated for me to understand and more complicated to explain to you so you can use it as a reference and then this part is also kind of these are all the amino acids and these proteins you can see this kind of shape and then they link together this fluorophore formation is one limitation of green it is slow rate of fluorescence acquisition in vivo I am sure you know in vivo and in vitro you know these type of terminologies what is that in vivo inside the in vitro outside in a dish yeah so so basically this is the process they are different proteins which after oxidize they try to attach with each other and then finally the final fluorophore contains a period of conjectured double bond so this is a kind of fluorophore which can be injected or that can be that can be available in this kind of jalyphation so this is a kind of being fluorescent protein so the biggest advantage of clear fluorescence protein is that it can be heritable that is next generation can have if the parent have this green fluorescent protein it can be transmitted to the next generation depending on how it was introduced allowing for continued study of cell and tissue so if you are studying a mutation or evolution of some living organism you can for inject a green fluorescent protein it will reside in the genome of parent cells and then it will move to the offspring and you can continually study these cells and tissue visualizing green fluorescent protein is non-invasive it does not change much to your proteins and because mostly you need invisible part apart from that some of UV range and requiring only illumination with blue light so GFP alone does not interfere with biological process so if green fluorescent protein is injected as a marker in any organisms they do not interfere with biological processes but when fused to proteins of interest careful design of linker is required to maintain in such a manner that they will not destroy that protein so then its applications are fluorescence macroscopy the availability of green fluorescence protein and its derivative have thoroughly redefined fluorescence macroscopy and the way it is used in cell biology and other biological disciplines while most small fluorescent molecules such as FITC this is fluorescent iso what is that I do not know or strongly phototoxic when used in life cells and fluorescent proteins such as green fluorescent proteins are usually much less harmful when illuminated in living cells so this has triggered the development of highly automatic living cell fluorescence macroscopy system which can be used to observe cells over time expressing one or more protein stacked with the fluorescent protein its basically an imaging technique you mark any cell or tissue with this green fluorescent protein and then you keep track of the changes happening in that living cells or tissue so this poor mice these two have green fluorescent protein middle one is normal so mice expressing under UV light so we human are really really very cruel so for these mice we are using UV light but for us we prefer that it should be blue light so these both compare to the normal mouse in the center then these are now it is a kind of fun for many big companies and industries Alba is a green fluorescent rabbit was created by a French majority using green fluorescent protein for the purpose of art and social so it's poor look at this poor rabbit it's really I feel bad and sad for it the US company Yorktown technologies market to aquarium shop green fluorescent zebra fish so that is again a fun these fish with this green fluorescent protein that were initially developed to detect pollution in waterway so now they are selling it so people are sure I want a protein fish or a rabbit or a mice so this neon pets as US based company has marked green fluorescent mice to the pet industry as neon mice so it's again a kind of pet you can have in your home for your kids and you can enjoy this okay thank you so much it was a kind of introduction to green fluorescence protein which can be has many application but it's not my feel you are like that