now there are two types of microscopy one of them is light microscopy and the other is electron microscopy and what is the difference between them so basically the difference is that what source of radiation we are using I'm K trees go as a source ofum as a source of Illumination use which we are projecting onto our specimen is it a light spectrum or is it an electron beam and a light and why do we need two different types of microscopy or um what is the benefici what is the pro and what is the con of both these microscopy we'll just discuss that in a minute actually further uh electron microscopy has two types which are um scanning electron microscopy and trans electron microscopy sub types of microscopy images or specimens so that you able to distinguish between a photo micrograph so basically light microscope through beic and what do I mean by this photo means light and micrograph is the picture obtained through microscopy and electron microscop micr electon micrograph okay now I have um in your notes I have mentioned light microscope or electron mosc function it is not part of your syllabus you do not have to know light microscope or electron microscope features lens um it's just extra content you do not have to know it I am just going to help you guys although P3 I am not going to be facilitating you guys with that because obvious have experiments onl but I am just going I'll just explain or give you a quick overview so basically in front of you you have a diagram of a of a light microscope microscopy so over here right over here we have a source of radiation ST which holds this speci in place now over here on the stage I will place my specimen my slide and say light so my light would be passing through this speci my specimen just a second right so my specimen is over here so the first lens that is present is our condenser lens diing and what we have to do is we have to focus all those Rays onto our specimen so that specimen so for that it is very important that our Divergent rays are converged and completely focused onto what on onto our slide so condenser l condens l focus and then the light rays would pass through these lenses lenses and objective lenses are the first magnifying lens why are we calling them our first magnifying lens because condenser lens it was not magnifying our specimen the sole purpose which it was serving was to project or Focus all the light RS on the objective lens magy and you have three or two or four types of objective lens in every microscope or turn different different objective l 4 time 10 and time 40 magnification so we have different objective lenses and you can rotate and observe your specimen through these three different levels of magnification then once it has passed through our objective lens the light rays and L is your secondy l so now for example light first magnifying lens objective lens power for example times 403 or IO magnifying power what times 10 so my total magnification is what my total magnification is both the magnification of my ipce and the magnification of my objective lens which means that times 40 and times 10 combine together to give me what 40 into 10 gives me 400 so my total magnification is 400 so what you have to notice that to work out total magnification you would be multiplying the magnification of both the lenses so for example if the magnification of objective lens is times 40 and the magnification of the ipce lens is times 10 you would be multiplying those two values and then you will obtain your total magnification of the light microscope okay then you have a CO knob and you have coar adjustment knob and a fine adjustment knob course adjustment knob stage optim between objective l l or Focus so that optimal distance the best possible distance between your slide and your objective lens is achieved and quanti enhance the clarity or the sharpness of your specimen fine adjustment KN so this is basically just an overview of how light microscope works it's not part of your syllabus it's just to just an extra knowledge so that now what you have to know source of radiation microscope obviously light rays maximum useful magnification so the maximum magnification that a light microscope can do the maximum or the maximum Factor up to which it can enlarge or magnify your image is times 1500 that is the maximum useful magic so the maximum useful magnification of a light microscope is times 1500 now the CCH over here is that a light microscope can view live and dead specimen y for example living and our light microscope produces colored images it produces color and it has two reasons why combin spectum so light is a combination of colors so reason why colored images are produced and the second reason is that sometimes what we do is we dye our specimen we use organic dyes to dye our specimen so that when we observing our microscope slide we can see colored um images so that have Z Clarity because black and white may be Clarity so organic dyes are used for staining to produce colored images and because light is a spectrum of vibor that is why light microscope is giving you um colored images then magnifying lenses are made up of optical gles made of glass then what are the benefits of light microscope light microscope portable that you can carry this light microscope from one place to another very easily it's not very difficult to transport it then it is inexpensive Capital it is easy to operate or use SCH levels High School levels College levels students are operating this and it is not very hard to operate and you do not need um skilled professionals like students and obiously microbiologist so they do not have to be very skilled to operate a light microscope light microscope is portable it is inexpensive so benefits now um let me show you a picture of a light microscope Che cells Che cells under a light microscope now if you could notice You observe cell let's focus on this cell only this is one cell basically tissue and you can only notice the outline of cells so usually light microscope detailed in depth organ information now moving to electron microscope now over here electron microscope a source of radiation that is El say Electron Beam now when I talk about electron microscope electron microscopes they are giving us a maximum useful magnification of what a maximum useful magnification of times 2500 250,000 y That's 2.5 lakhs so useful magnification in light microscope that was what that was just 1500 um multiply by 1500 and in electron microscope VES 250,000 magnified h now drawback electron microscope that it only gives us we can only view dead specimen through it and why do we view dead specimen through it just in a while AA okay now images produced are black and white a light microscope was giving us colored images on the contrary an electron microscope is giving us black and white images metal stains are used for acquiring a better contrast of color basically electron microscope strictly black to contrast basically difference in the color of black colors gray darker black whitish black contrast basically distinguishing clearer view electromagnetic lenses are used why I just get to you in a bit microscope electron microscope multip by 250,000 times over here this is a structure of a mitochondria now mitochondria is present inside of a cell proper and subsequently mitochondria 70s ribosomes so not only are you able to see a mitochondria you're also able to see what is inside a mitochondria through an electron micrograph so obviously electron micrograph magnification and it is very beneficial but why is it can only VI dead specimen then it is very expensive and it is not portable then it requires skilled expertise to operate and so large set professional expensive overall total capital cost to use an electron microscope that is very high so just because it's very expensive because of the labor required because of its own Capital price because of space requirement de speci speci and why is that it is because let's understand so elect El this is entire chamber liquid solid this is a vacuum ch okay it's a vacuum chamber and can anybody of you suggest why are we creating this vacuum light microscope what is the need to create a vacuum why can not a medium be present in a chamber of electron microscope what do you guys think okay so now Beam Beam like if I tell you the mass of an electron that is 9 into 10 the power 9.1 into 10 the power minus 31 K that is how small an electron is electrons they're very tiny particles molecules present liquid molecules basically [Music] specimen so we are creating a vacuum to ensure that the Electron Beam does not collide with air molecules and get scattered a scattered Electron Beam produces an unclear image why because it is not focused or Focus proper magnification how does that give an answer to the fact dead specimen because specimen live speci cylas now cytoplasm is a fluid it is a gel so obviously water content fluid content so what happens is that in vacuum if I keep a cell a live cell that water basically boils in vacuum or M Water so obviously my specimen is destroyed it is dehydrated it is dead so I do not want my specimen to be ruptured so that is why I'm using a dead specimen or dead specimen dehydrate water content and then we keep it in that vacuum chamber so that it is not destroyed or it does not undergo leses is it clear so far any questions now let's quickly understand transmission electron microscope or scanning electron microscope differ so transmission or scanning it's very much suggestive in their name specimen draw um blue material that is basically my specimen this is our specimen yes SL now what happens is that the entire electron being it transmits through that cross it is getting transmitted through the entirety of that specimen now for this the electron the specimen has to be extremely thin why because El beam to completely pass through it and obviously prep but the kind of image which is produced through this is a 2D image so transmission electr one that the Electron Beam is completely passed through it and second that it is producing 2D images now this is something the 2D image while point this is something you must remember for your exams because exams transmission micrograph or scanning microgap and they ask you to distinguish between them draw talk about the differences and the similarities so the first and foremost observable difference that you would notice about a transmission micrograph sorry a transmission electron micrograph that would be that it is producing 2D images now when we come to a scanning electron micrograph what happens is that our Electron Beam reaches the surface of the speci and it gets reflected or reflected beam that is basically captured on the screen or is deflected be and because elic that would be a 3D image and scanning electron micrograph is responsible for providing us 3D images so now I will basically be showing you the differences now if you look at a slide of a light microscope light microscope and we were only observing nule usually mostly now this is a trans Mission electron micrograph this is a scanning electron micrograph a mind you I just said electron microG black and white now you would ask me scanning electr micr so what happens is obviously computered system so through technology what we do is picture screen computer and white computer colors so now if you could see this is a 2D image mic scanning elect mic of that can You observe that can you appreciate that are you able to distinguish between a t and a electron micrograph different right okay so that is all for microscopy let's quickly do resolution and we will wrap it up for today AA ABA you were not here in the start of the class so the things that I have discussed um I'll send you a message on PM so you can see but one thing I want to let you know that for tomorrow's class I will not be taking that class and we have decided a time for an extra class on Saturday so U I talked with hadra and I talked with musan and they had suggested that we keep that class at 12:00 p.m. or 12 noon on Saturday so would that work for you are you fine with that otherwise if you're for some reason busy then you can obviously access the recording and I have discussed with uh these people so I'll just send you a voice message about that once the class is over now moving on to resolution for that let me show you something so um I'll just remove the blur okay now these are two papers up exactly identical Pap now with this distance between them you can easily tell me that this is one paper and this is the other paper and are clearly distinguish between these two people between these two papers you can clearly state that this is one distinct object and this is one distinct object now if I overlap them now if I overlapping them now these are overlapped now can you tell me you a paper that distinguishing factor that is no longer noticeable you cannot tell me if this is one paper or if this is two paper but if I increase that distance between these two papers you can clearly state that these are two papers that is exactly what resolution is resolution is basically your ability to distinguish or to see two separate entities to separate points as distinct points for example this is one point this is another point right now you can tell me you don't same points like they don't doal points and this is one point and this is the other point now if I bring it here now can you tell me if these are two points or if this is just one single point or one single entity now you cannot tell me that are these two points point and why is it so I was able to view this as two distinguish as two distinct objects as two distinct points because of the distance between them that was the same reason why I was able to see these two papers as two different papers as two separate papers why because of the distance between them when I close this distance and I bring them like this they become one point clearly distinct points distinct objects yeah a object that is what resolution does resolution is defined as the minimum distance between two points before they can be seen as separate points Yan let's take this paper for you to understand or for you to identify these two papers as distinct papers minimum this is the minimum distance at which you can say that these are two distinct papers but minimum distance now you cannot tell me if there are the same paper or if they are two different papers when we viewing a cell under a microscope We There is a limit if they are at that minimum distance then I can see those two organ those two substances as two separate points I can distinguish them as two distinct points but if I narrow that down I will not be able to figure it out so that is what resolution is resolution is the ability to distinguish between two distinct points and on what factor does resolution depends electron mag microscope or light microscope and we said that light and Electron Beam there are two sources of radiation okay now what happens is that light wavelength and when you will come in A2 and you will be studying physics in A2 you would understand that electrons also have a wavelength when they're behaving as a beam when they're behaving as a wave so electrons have a wavelength and our light obviously has a wavelength light wavelength that is 700 to 400 nanom so basically when you are moving from wavelength of change so minimum wavelength n resolution the magnet ude of resolution is almost half of your wavelength so Lighty wavelength 400 nanom 400 nanom 200 nanometers that is my resolution for a light microscope and what do I mean by that two objects have to be 200 nanometers apart for me to see them as two different objects under a light microscope which means this is one structure structure structure and they have a distance of 100 nanom when I'm observing this under a light microscope would I be able to see these two structures as one point or as two separate points light microscope minimum distance between two points 200 nanom for me to observe those two substances those two particles those two organal as two distinct two separate points now these are 100 nanometers apart 100 nanometers is less than 200 200 100 Nom now I cannot tell these two objects apart so there should be a minimum distance of 200 nanom between two points for me to distinguish those two points as distinct or separate points is this clear or do you want me to repeat it a now for electron microscope now elect micc now just observe or differ 100 or 1 nanom differ electrons have a shorter wavelength and 1 nanom half one half I would get I would get5 nanometers and now for5 nanometers now tell me is 200 nanometers a greater value or a05 nanometers is a greater value obviously 200 nanom distance 200 nanom for example or Nom so now when light microscope they should be at least 200 nanometers or more apart from one another for me to observe them as two separate points in a light microscope but in an electron microscope m pass5 n minim now n which is greater than 0.5 .5 nanometers say distance so I can easily observe that thing now tell me what is the resolution of light microscope better or electron microscope what do you think 200 nanom resolution better nanom res solution so basically in an electron microscope Nim separate points so resolution resolution nom distance a come value from 200 nanom distance but me light microscope a. n n Nar electron microscope 100 nanom clearly so for me a lesser value of resolution is a good value and if you could see light microscope wav 400 nanom value of resolution on the contrary in in an electron micc El but this wavelength is giving me a better resolution so I would say that shorter the wavelength lesser the wavelength higher is the resolution of my microscope unclear resolution low so higher the resolution more clear is my picture so magnification enlarge or resolution basically is responsible for the clarity of the image so that's it for magnification and resolution