hello guys. In this video, I'm going to speak about a very important or very very interesting mechanism called the flow cytometer. In fact, actually, I did two videos, and in this video, I'm going to mention specifically flow cytometer, and in the next video, I'm going to mention the facts mechanism fluorescence-activated cell sorting. Let's start by speaking about flow cytometer. Flow cytometer is a cell counting mechanism, so it's used to count the cells and together to analyze the phenotype and the health situation of the cell. How does it work in general? First, in general, in flow cytometer, I have something called the flow cell. In the flow cell, we add our cell mixture. Then I have the sheath fluids. The sheath fluids will apply something called the hydrodynamic focusing. The hydrodynamic focusing, or the sheath fluids, will send the cells one by one through the flow cytometer tube. It's very important to pass the cells one by one through the tube because here I have something called the laser beam, and this

the cells should pass through the Laser laser beam one by one. This point, where when where the cells pass through the laser beam, is called the interrogation point. Now, when the cell pass through the laser beam, there are two events happening. First, if the forward scattering, which is the light scattered in an acute angle to the forward direction, and the side scattering glass, which is the light scattered in unison in every other direction in side directions. These two events or this light scatter scattering is detected by specific detectors called the forward detector and sight detectors, and these detectors are connected to a PC or a data analysis and aisle analyzer, which is going to analyze the data obtained from the detectors. Now, this is in general. Now I'm going to speak in more in details, saying that this is the sat flow cytometer of flow cytometry tube, and then the cells pass one by one through the laser beam. When the cell paths through the laser beam, the cells scattered the light in a forward direction, which is in a

light scattered in an acute angle. This forward scattered light is directly proportional to the size of the cell. Why? Because the cells scatter a particular amount of light in the forward direction. But when the cell is bigger, the forward scattered light will be larger, or like yeah, more and more scattered light will be detected. And when the cell is smaller, the cell will scatter less light in the forward direction. So, the forward scattered light is directly proportional to the size of the cell. Now, the detector detects the pulse of photons which are the light scatters. The forward scattered light is a pulse of photons which are detected by the detector. And the detector then, as I told you before, is connected to a PC or computer. The PC will convert the intensity of the light detected by the detector into voltage. And then, I will get a specific amount of voltage for every cell passing through the laser beam. The PC will give me data similar to this. So, every time a cell passes through the laser beam, I will get a

specific peak like high voltage peak for large cells and low voltage peaks for small cells. The data or the forward scattered light data at the end be similar to this time versus voltage, and then the peak will be proportional to the size of the cell. This is forward scattered light. The second events happening in the full earth flow cytometer is the side scattered light. So, awareness when a cell paths through the laser beam, the cell will scatter the light in side direction, and this side scattered light is directly proportional to the complexity on granulizer, the granularity of the cell. Why? Because the simple cells scattered light in the side direction, yes, but when we have granules inside the cell, so when the cell is complex from inside, these little granules will also scatter light in different directions, and in this case, I will get more side scattered light like this. So, this is a complex cell from inside with it contains small granules inside, and then these granules will also scatter the light in different directions, and in this case, I would get

a higher intensity of the site scattered light similarly to what we saw in forward scattered light. The detector, our detectors, are connected to the PC, which is going to convert the intensity of the light into voltage, and then I'll get something similar to this. So the side scattered like that I will be similar to the forward scattered light data. So for the complex cells will give me a high peak, and the simple cells will give me a small peak. Now, in order to analyze the cells, what I have to do is that I have I can do I can combine both forward scattered light and side scatter light data together. In this case, I'll get something called the 2-dimensional histogram, the 2-dimensional histogram. Its name, it's two-dimensional, so I have side scatter and forward scatter data all together. In this case, where I get is something similar to this. So I have here four quadrant. If the cell appear in the lower quadrants, it means that it has low signal on the side scatter detectors, which means that the cell is simple if

the cell appears in the upper in the upper half. It means that it gives a high signal or the slight scatter detectors. So, it means that the cell is complex. If the cell appear on the right half, it means that the cell give high signal on the forward scatter, which means that the cell is big. And if the cell appears here, it means that the cell is small. So, if the cell appears, for example, in this quadrant, it means that the cell is small and it's simple. If the cell appears here, it means that the cell is small but it's complex. Here, it means that the cell is big and complex. And here, it means that the cell is big but simple. I will give you a very good example of this, which I have an example about the analysis of a sample of white blood cells. So, what we see here is an analysis of white blood cells. This is like scatter and this forward scatter light. What we see here is a group of cells which are complex, very cupric complex cells because they give a high signal on the side scatter.

detector and they are medium inside. They are not so big, and they are not small. So, there are Windom inside. Obviously, these cells are granulocytes because granulocytes are like, as in a few buzzer, few of neutrophils. They are medium inside. They are very complex cells. Here, I have bigger cells because they give almost like they give more or less high signal on the forward scatter, but they give low signal on the side scattered. So, they are big, but they are not too complex. They're simple cells, and these are monocytes because monocytes are big cells, but they are not complex. Here, we have very simple cells because they give very low signal on the side scatter, and they are like smaller than monocytes and granulocytes. So, obviously, these are lymphocytes because the lymphocytes are smaller in size, and they are simple cells. Of course, this is a very useful technique used to count the cells and to analyze the phenotype and the health situation of the cells, and what's very

special about this technique is that we can analyze many thousands of particles and cells per second. It's like it's an easy technique. It's not very hard to do to perform, and it's precise. It's very used nowadays in all the laboratories. This is everything I wanted to tell about flow cytometer, which is very important to know. If you want to know more about facts mechanism, in the next video I would speak about fire effects mechanism. So if you know you want to know more about facts mechanism, then you want to, you need to watch the next video. If you liked this video, please don't forget to Like, share, subscribe to the channel, and if you have any questions, leave it in the comments. I will answer you and see you in the next video. Bye.

The Principle of Flow Cytometry and FACS (1- Flow Cytometry)

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