 Learning objectives include what is light microscopy, how a light microscope works, what are various parts of a microscope, and what is total magnification resolution and refractive index. A light microscope is not light because it does not weigh much. It is because it uses light as a source of illumination and that is the reason we call it a light microscope. As you can see here, there are various parts. This is the stage of the base where the rest of all these parts are lying. Here is the illumination source, which is the light source. And this is a stage on which we put a slider specimen. Then this part is the objective lens. There is a host of three, four different lenses with different powers of magnifications. This part is called the eyepiece and here is a knob for focusing the image. Now the basic principle which is used for making a microscope is this, that this is a lens which is a convex lens. If there is an image or an object in this case, if it lies outside the focal point, you can trace where it would form the image, like here. This is a candle which is, this part here is the focal point of this lens on this side as well as on this side here, the focal point. So the light rays, this is a ray diagram. You can draw a light ray with parallel to the long axis or the main axis here. And when it strikes the lens, it then bends and passes this light ray, would pass through the focal point. And then if you take another ray which passes, let's say, in the, through the middle of the lens, it does not get bent. It remains straight. So where these two meet, the image would be formed. And of course, this image, because it is formed by converging of these rays, it is called a real image. And it is upside down and maybe smaller, is much smaller. If the object, it lies within the focal point, or in other words, between the focal point and the lens, then you can draw the ray diagram. The ray diagram here, that this ray goes, which is parallel to the long main axis, it passes and gets bent and would pass through the focal point. And then another ray, light ray, would pass through the middle of the lens. And that does not get bent. So it remains straight. But if you trace them back here, these are kind of diverging rays. So they never would meet. But if you take these rays back from here onto the same side of the object, you see them meeting somewhere here, like here. So this image, which is on the same side of the object, is straight. But it is called a virtual image. And it is formed by the diverging, if you trace them back. So by the diverging rays, if they're traced back, a converging, the image which is formed by converging rays, as you saw here, this could be taken on the curtain or the screen. And that is why it is called a real image. This one is a virtual image. We cannot take that on a screen. We cannot project that onto a screen. The microscope basically utilizes this principle here. It is composed of two lenses. This is the objective lens, which basically makes the first image. So this star, which is our object here, you can trace with the diagram, the ray diagram, that the image is forming somewhere here. But look, for this lens, this image falls within the focal length of this image. And then it is formed by these diverging rays here. And that is the reason it is much bigger. And it is inverted, of course. So the image, the two lenses are aligned in such a way that the image formed by the first lens, it lies somewhere between the focal length of the lens, this lens, which is called the eyepiece here. So the image of this lens is created or is formed somewhere between the focal length and the lens. And that image, when you trace them back by these diverging rays, they converge on this side, the image becomes bigger and is also inverted. So this is the principle of a microscope. As I mentioned earlier, these are various parts. There is an illuminator, which is the source of light here to illuminate the specimen. Then there is a condenser that condenses all the light so that the image, the specimen is illuminated very well. There is the objective lens that gathers the first image. And then there is an eyepiece or ocular lens that basically perceives the image. Magnification of objective lens times magnification of ocular lens is the total magnification. So there is some magnification that occurs with the objective lens. And then there is some, and these magnification powers are written on the lenses. So objective lenses come in three different powers mostly. The low power is 10x, 10 times magnifications. There is a high power, which comes with the 40 times magnification. And there is a 100 times magnification lens, which is called oil immersion. We'll talk about this oil in a minute. Why we use this oil with 100x lens, objective lens. Ocular lens are mostly 10x. So with low magnification power, the image is 100x amplified or magnified. With oil immersion, it can be amplified 1,000 times. Now the term refractive index basically means that this is the ability of a medium to bend the light. So the light, when it passes from a dense medium into a less dense or thin medium, it goes away from its vertical point. Because this is the condenser, the light source, which is putting that light through this specimen. And when this light passes through the glass here, glass is a dense medium. But when it passes through the air, most of this light actually is bent and is what we call diffracted. And this lights go away from the objective lens. So objective lens cannot gather this light. In order to make this light available, what we do is we put an oil, what we call immersion oil here. The density of the oil or the refractive index of the oil is exactly like glass. So all the lights raised that are in here, they don't bend. They simply go into, they enter into the objective lens and illuminates the image much more. And it is easy to see the image, especially when we use 100X lens, we have to use immersion oil. And that is the philosophy of using, that we want to capture all those light rays that can go astray. Also in order to increase the contrast, we stain the specimen. So keep that in mind. Staining is basically to increase the contrast. So if there is a contrast, we can see, our eyes can see that organism or that object easily. Resolution is basically the ability of lenses to distinguish between two closely lying objects as separate. As you can see here, that there are two objects that seems lying almost overlapping with each other. So the resolution power is low of this microscope here. But if we have another microscope that can resolve them better, although they still overlap, so but of course we can see that these organisms, they overlap less. But here, we can see these objects as separate ones. And naturally, this microscope is the one that we would prefer. So the resolution power of a light microscope basically depends on if all these parameters are everything else is equal. This lambda, which is the wavelength of the light, we use light as a source of illumination. So it depends on the wavelength only. So shorter the wavelength, the bigger would be the resolution. With the microscope, the light microscope, we can resolve two organisms that are 0.2 micrometer apart or 200 nanometer apart. The refractive index of oil immersion that we used in the previous slide here, just for your information, it is 1.5. It is exactly the same as the glass. So all the light source, the rays that are there in that pass through the specimen, they do not go away. They all get entered into the objective lens and illuminate the specimen very well. In summary, ordinary light microscope uses light as a source of illumination. In order to increase the contrast we sometimes, but mostly with light microscope, we use stand objects and the resolution of a light microscope is only 200 nanometer. That means that if the organisms are 200 nanometer apart, we can visualize with the light microscope.