And then we have a super resolution imaging system, the super resolution imaging system for the traditional confocal microscopy. The fraction, the limit, of optical the fraction is around 200 nanometer. Meaning that anything smaller than 200 nanometer we cannot see or see clearly and there microscope because each fluorescent protein here make it looks like a dot. The image of each fluorescent protein particle looks like a dot, but actually the microscope it looks like a moth, not only a single dot. So with this resolution that we can now see clearly the things, the tiny things under this 200 nanometer. So people develop this technique called super resolution microscopy. Let's say we labelled things, labelled a structure in the cells, like these green lines. We still label them with fluorescent protein. In step one, we randomly activate certain dots, certain particles within this labelled flourescent structure. These yellow tiles or orange tiles are the ones we activate in the first round, in the first step. And then, with the computation that we can calculate the center point of each dot we just activate and then the computer can record this centerpoint of this dots. And on the second run, the second step, we still randomly activate, other fluorescent point, fluorescent dot molecules then again with the computation we can calculate the center point of the start and label the start and mark the start. And then we do this several rounds, third round, fourth round, several rounds. And eventually, we can mark all this dots together and then can get the image. So if we compare the traditional conventional image to a super resolution image we can see as showed here so the left one is confocal, traditional confocal. You can see the microtubule. This is the second scan, it's a microtubule structure within a cell. So you can see these microtubules they are blurred, they're not very clear. And then, with the computation of super resolution. Then we get this, these image after calculation. We can get a much clearer pictures with much higher resolutions. So, with storm, so now we have three techniques for the simple resolution image. One is called the parm, one is called the storm, and one is called the sim. So the principle of these three techniques are almost the same. There's a slight difference between the three techniques. So with storm now we can push the resolution, the optical. Now the optical resolution itself is calculated resolution to around 30 nanometers, and the idea or the dream of is to push this limit to five nanometers. But now it is still around the. So if we see this picture of more clear idea. So the top one is traditional confocal. So the microtubules and then some other protein label the red scattered in between. If you blow up this picture, it's just pixels, it's not clear at all. But, with the process of storm microscope, microscope that we can see images at much higher resolution than everything is more clear here. So the same thing for here. The top panels are confocal image, and then the bottom are storm image. So the storm image is much clearer than the traditional confocal microscopes. So if, with corpuscles, or with super resolution imaging, if you want to observe six samples, then what should we do? So we can use two photon images. So the two photon image can make it possible to observe, to see the six samples. So the two photon, the advantage of the two-photon is that longer wavelengths are less affected by the scattering than the short wavelengths and easier pass through the samples. So that we can observe our samples in does a six samples. And then the fluorescents, the fluorescent proteins or the molecules outside the [INAUDIBLE] place are not excited, and more exciting light reaches the focal plane, and penetrates to the deeper samples. So that we can use this one to the [INAUDIBLE] the six samples. The other way, the limit of two-photon imaging is about micrometers, a couple of micrometers. If we want to observe brain tissues in micrometer range then we can use two-photons. If we want to observe we want to see the hovering or the tissues or the samples much thicker than micrometer scale. So, what should we do? We have this so called MOST technique, the full name for this MOST, micro optical sectioning tomographic. A simple. A machine to catch the point or to catch the samples into pieces, into slices and all the cup of this brain cutter we have lens of the microscope. So with the cutting, with the laser cutting slices and then the microscope can take image of the slice. And so the laser cut and then the microscope takes the image of this thin slice. So theoretically, you can observe the whole animal brain by cutting a low microscope, right? But the first generation of them most is unfortunately it's very slow. To observe the whole [INAUDIBLE] the [INAUDIBLE] mouse brain, will spend, you can guess? We'll spend how much time, we observed whole mouse brain. Well that's the whole process, it's very slow. And now, the good news is now we have the second generation, the third generation of the MOST. With the third generation of MOST, we can cut a MOST spring within a week. Yeah, it was one week that you have the picture or the image of the whole mouse brain and the fourth generation is coming and they say for the fourth generation, we probably we can have a whole mouse brain cut and imaged within one day. That's acceptable, right? So the of using this technique is the connections of neurons with certain are. So this is an example, a 3D example. A 3D image example showing the neurons and the connections with one sick tissue, within one sick tissue. And this is another fancy example showing the printer types neuron located and connected in one big brain area, in one big sick sample. And then if you don't want to wait for one month or one week for to cut and take picture, you can have this scale. I don't know, I don't even know the Chinese translation for this scale. Scale is a technique published several years ago, developed by a Japanese crew that you can soaked tissue that you want to or the embryo into a certain solutions. And the after one month of soaking, you take it out, it become transparent. the whole thing become transparent then you can do the labeling and the microscope because this is transparent tissues. The only disadvantage of this so called scale technique is that your tissue becomes bigger than the original ones you can see from this picture, it's becomes bigger. But, according to the original paper published by this Japanese group, although your tissue, your sample becomes bigger, it's not distort. So every part of the body, of the tissue become bigger at the same rate, at least this is what they claim, right? So this technique is still usable for observe sick tissues, and then the cells if you want to observe the cells underneath your tissue underneath the layer. Electron microscope, if you want to see things smaller than super-resolution imager can give you, then we can have electron microscope. Electron microscope is different than our optical microscope. Basically it shoots the sample this electron. And then with the reflection of the electron, then we can know the shape or the side structure of the sample.
