hello and welcome to learn a level biology for free with mr. ik today we're gonna be going through microscopes and this is one of the topics that I frequently get requests in my tutoring to have help with going through the magnification calculation the conversion of units and mainly the calibration of the eyepiece grass cue so we'll be covering all of that in today's session so if you want get some paper at the ready for some of the questions and make notes as we go so this all falls under topic 2 within the methods of studying cells and once we've gone through the cells it's knowing how scientists discovered that these are the internal structures our cells have and this has been worked out through using microscopes and self fractionation and ultra Gatien now this video we're just going to be focusing on the types of microscopes magnification and calibration of the eyepiece graticule and but for the link to the next video on self fractionation and ultra centrifugation that'll be at the end of this one so you can just click the link to that one say microscopes this is something that you did cover at GCSE and there are three key types of microscopes you have the optical or light microscope and the electron microscopes but there's two types of electron microscope you have a transmission or a scanning electron microscope now the first bit of recap to have a go at is can you still remember from GCSE the definition of what magnification means and resolution or resolving power so pause the video at this point to see can you come up with a definition for those to write the definition for magnification is the magnification of the microscope is referring to how many times larger the image that you look at is compared to the actual size of the object the resolution of a microscope is the minimum distance between two objects which they can still be viewed as separates so by that we mean if you are looking at your sample through the eyepiece under the microscope the minimum distance in which you can still see two different parts of the cell has been separate rather than then they have just blurred into look like one single point so the resolution or the resolving power in an optical microscope is determined by the wavelength of light and in an electron microscope it's the same idea it's the wavelength of the beam of electrons so let's have a look at these microscopes in a bit more detail at this point I've just split it into the two types generically so the optical microscope will be the type of microscope you're familiar with this is what schools and colleges will have access to and in this case you have a beam of light being released by your lamp or sometimes it's a mirror to reflect a plugged in lamp and that light then gets shown up and is condensed and that is what creates the image now you have a much lower resolution on light microscopes compared to electron microscopes because the light has a longer wavelength and that is what determines the resolution of microscopes it also has a lower magnification in comparison but you can get color images so what you're viewing you will see those real colors and you can use living samples in contrast the electron microscope whether it's scanning or transmission for both of these this time the source is going to be missing a beam of electrons and those will be condensed using electromagnets and that is what is going to create the image electrons have a much shorter wavelength so you get a higher resolution or resolving power higher magnification but you can only get black and white images and the samples that you've you have to be in a vacuum so completely vacuum no air and for that reason you can't view living sample so a bit more about the optical microscope we've talked about in fact it has a lower resolution because of that longer wavelength what that means in terms of its application is you can't see the inside of organelles in detail and some of the small organelles you can't actually see at all so I've got an example over here where we're looking at mitosis in root tips and you can see that there is an outer layer you can't actually see the difference between the cell membrane and the cell wall we know that this liquid in the middle must be the cytoplasm and stained purple are the chromosomes so we can see where the nucleus is but that is the level of detail you can see with the magnification and resolving power of an optical microscope the electron microscopes so going back to what I was saying the specimen has to be in a vacuum and the reason for that is the electron beam that is released those electrons would be absorbed by the air and never even reach the sample or specimen to create the image so for that reason has to be prepared as a vacuum and that's why you can't view living samples and you only get black and white images so a bit more detail the transmission electron microscope so transmission means to pass through and that is what is happening in a transmission electron microscope that beam of electrons some will be absorbed by the specimen some will pass through and that's why you get these different shades of white and the black and the darker is the more electrons have been absorbed and that's how you get this detailed image below so it's a 2d image it's only black and white but you can see this here is our chloroplasts inside of a plant cell and you can see the internal structures you can see these thylakoid membranes you can see the grana those stacks of the membranes as well scanning electron microscope you don't have to have these very very thin samples like with the transmission this time it's not going to be transmitting so passing through the specimen instead it will scatter and reflect off the surface and because of the different depths of the specimen that will affect the scattering of the electrons and that then creates this 3d image so the scanning electron microscope will give you details on the image to do with the texture and the 3d depths of either your cells or your organelles so moving on then magnification is one of the math skills linked to microscopes and it's used with optical microscope images so the formula again straight from GCSE image size equals actual size times magnification now I've deliberately written it this way rather then is the magnification formula because I think this is the easiest way to remember it I am so I beam image a is the actual M is the magnification and then once you can remember I am you can rearrange the formula to work out magnification or actual size now that is a skill from GCSE maths but if you can't remember that I'll link up the top here just click to see one of the GCSE videos I have on rearranging the formula just to recap to get your aid of a math skills up scratch so the other thing that you'll need to be able to do is one of the math skills is converting units and that's because your image size so this is when you're going to be measuring your microscope image which is also called a micrograph you'll be using a millimeter ruler so your image size will be recorded in millimeters the actual size of cells and organelles is much smaller it's going to be micrometers and in order to use this formula you have to have both of those sizes in the same unit so if you have recorded your image size in millimeter then you'll need to convert it into micrometers so you have the same units to go from millimeters to micrometers you multiply however many millimeters you have by a thousand so if you measured two millimeters that means you have two thousand micrometers all you could do the conversion the other way round if your actual size is in micrometers you can convert your image size into micrometers as well and in which case if you had two thousand micrometers to convert that back into millimeters they'd be divided by a thousand and you have two millimeters so just to go through a worked example we've got here it's a bit blurry but I'll put in all the details you could be asked to work out what is the magnification of this micrograph image and they've given you a scale bar and if you've shown a scale bar what that means it is the length of that bar is representing an actual size of 50 micrometers on this image so our scale bar is the actual size and that has 50 micrometers what you then need to do to work out the magnification we noted need to know the image size and you don't need to measure any cells for this you are measuring what is the image size of that scale bar so you'd line up your ruler and measure how many millimeters long your scale bar is and in this example it's 20 millimeters and I measured it so now we know we'd be doing our image size which is 20 millimeters divided by our actual size of the scale bar which is 50 micrometers but we need to get those into the same units so I'm going to convert 20 millimeters into the micrometer units to match the scale bar so I need to do 20 times a thousand so that gives me 20,000 that is our image size divided by our actual size is 50 so our magnification of this image is 400 times magnification so finally the last skill is the use of an eyepiece graticule and how you calibrate it so I'm just going to show you this image here of the microscope first of all just so you can see where the eyepiece graticule is located so this eyepiece graticule is a glass disk which is within the eyepiece and that glass disc has a scale scratched or etched onto it and that is so you can line it up on top of whatever you're visualizing to see how many divisions on your eyepiece graticule does the nucleus cover for example and that can then be used to measure the size the actual size of the objects that you're viewing under the microscope rather than using the formula that we saw on the previous slide however it's not quite as straightforward as that because as you're using your light microscope you will be potentially moving between these different objective lenses and each lens is a different magnification and what that means is the divisions on this eyepiece graticule scale will be worth different distances depending on how magnified the image is and that's why we have to calibrate the eyepiece graticule each time we use it at a new magnification so to calibrate it you need to use a second scale which is called a stage micrometer and this is a glass slide looks quite like a glass microscope sight and it's called a stage micrometer because this is the scale that you're place on the stage and it's measuring distances in micrometers so the scale on it now mine isn't quite to scale but that scale that you have scratched onto this piece of glass is two millimeters long and each single division is worth 10 micrometers now I've only done every 10 divisions on this that's all I could fit in on the diagram so one division is worth 10 micro meters so 10 of these divisions is 100 micrometers long so if we go through step by step then how you would use the stage micrometer and the eyepiece graticule to calibrate the graticule it's a step 1 you'd place your stage micrometer on the stage look through your eyepiece and this is what you should see you have the eyepiece graticule scale and then line that up directly next to your stage micrometer scale so step 1 line them up so they're next to each other as you can see in this image step 2 you need to count how many divisions on the eyepiece graticule scale fit into one division on your micrometer scale now you might find that easier to work out how I'm doing in this world example we can see here that we have 20 divisions from the eyepiece graticule scale fit into 10 divisions on the stage micrometer scale so I've got 20 fitting into 10 or in other words 2 of the divisions from the eyepiece graticule scale fit into 1 division of the stage micrometer scale so we have a ratio of 2 divisions to 1 so now we've got that we can link it back to what we said about stage micrometers on our stage micrometer 1 division is always worth 10 micrometers so you can use that to then work out at this magnification what is one division worth on the eyepiece graticule so we said one division on the micrometer scale is 10 micrometers we said two divisions fit into one on the micrometer and if one division is worth 10 but to fit in each time is 10 divided by 2 and we know then that on the eyepiece graticule one division is worth 5 micrometers at the current magnification so now you can take out your stage micrometer put in whatever slide you want to use to measure the distances or size of some of the cells organelles so I've gone back to the slide that we saw earlier on and in this case I'm going to measure the distance or the length of the nucleus in this example now you would have all of the subdivisions I've just not shown it in this image so where we've got 40 to 50 50 to 60 you would have an extra 10 divisions so you could see 41 42 43 and so on so we've said that we've worked out that our eyepiece graticule at this magnification 1 division is worth 5 micrometers so I'm now going to measure to see how many divisions the nucleus covers and I'm estimating that is 13 divisions we've got 10 and there may be 3 more so the nucleus is 13 divisions long 1 division is worth 15 so multiply that by our 5 that's 1 divisions worth 5 and 13 divisions so that means in total the nucleus actual size this magnification we've worked out it's 65 micrometers now it will be that distance or length even at every magnification the only thing that would change is what one division on the eyepiece graticule is worth at different magnifications so that's it for microscopes so just to go over again a summary of some of the key points you need to know you need to say the differences between optical and electron microscopes and the electron microscope has a much higher resolving power and magnification and that's why the electron microscopes can be used to see the details inside of organelles the formula I am so image size equals actual size times magnification can be used to work out the actual size of a structure or the magnification from your microscope image and lastly the actual size of structures can be measured using the microscope if you have an eyepiece graticule in the eyepiece but you have to calibrate it first with the stage micrometer it's take into account the different magnifications and that's it for microscopes for a level bulging now there are quite a lot of linking videos to this so if you want to just go over some of the content or the math skills over here are linked to the rearranging the formula as well as you've got your eukaryotic cells and prokaryotic cell structures and last if you aren't subscribed already click the logo here to subscribe to make sure you don't miss out on any of the latest videos