So basically if this is this flake, this snowflake, is your sample, you shoot this sample with electrons. And then the electron reaches the sample, hits your sample, and then be bounced back. Then the receiver will receive the bounced back electrons and we'll read them. And then with the reflection rate or speed of your electron, then they will calculate, the shape and then the structure of your sample. So this is another image taken by the electron microscope. What's the? It's the electron microscope. We have two kinds of. One is transmission. So for the transmission, by name we know the electron has to pass through the sample. So, this is an example of transmission. So we have to make our sample very, very safe to let electron to pass through. So, where the electron can pass through, where there's no material or no substance, the electron can easily pass through. When there's material or there's very compact material, the electron cannot easily pass through or cannot pass through at all. So for example, this cell or this compartment where there's lots of stuff there, where lots of materials around to form a very compact structure. The electron cannot pass through. So showing the very dark color, almost black color here. So where there's less materials, will be some electrons can pass through, so showing the gray color here. Where there's no material, like this blank region here, the electron can pass through almost 100%, so that's empty space. This is a transmission Showing the cellular structure within a neuron, so this big part here is the nucleus of neuron. So this structure here are mitochondria. This big trunk here is dendrite. This structure of mitochondria, and then this dark dot here is lysosome. So from this picture we can know lysosome is the most dense compartment in the cell. And then after that is the mitochondria, and then after that is the nucleus. So, nucleus has the most light color within the cell. And then this picture here, is what we call a synapse. The synapse between two synapse, there's what we call the dense area. Postsynaptic [INAUDIBLE] with very black staining, or not staining, image in the Picture. And then all these dense area showing one synapse. So there are several synapse in this picture. And this is a transmission Showing a Golgi apparatus. So this is a very typical Golgi, looks like in our textbook. So transmission Can only label physical structures. Cannot tell us a specific location of specific proteins. If we want to know the locations of specific protein we can do immuno. The immuno Is when we use the antibody to specific proteins. But the antibody do not have any density or color or anything, so we link the antibodies, specific antibody, with gold particle. So the gold is a very dense particle, so the electron cannot pass gold. So in the immuno Here, whenever the antibody labeled protein will show black dots here. Because the gold particle will prevent the electron from passing through. So, the black dots are basically where your proteins are. And then with this technique we can label up to two or three kind of different kind of protein in a single sample. We can use small gold particle or big gold particle. Sometimes even people push this harder, use middle size particle. But that's really the small size and the big size are more easier to see. The middle size, people always argue with the middle size, right? So, these are the examples of immunolabeling That you can see that the black dots are where your protein labeled. And then here's another example of the synaptic proteins are labelled by immuno. So compared to the transmission Where the electron has to pass through the sample to make an image, we have another kind of Scanning. The scan We only can see the surface shape or the structure of the sample. The electron do not pass through the samples. They reach, or they hit, the surface of the sample, and then they bounce back and then because of by the scanner, by the receiver. So the scanning Can only describe a surface shape or structure of their sample. So these are cells, described by the, imaged by the. So there are some, although the EMs the transmission And the scanning Can help us to see the tiny structures in detail of your samples. These are the advantage of the. One is the sample treatment. For both scanning and transmission The sample treatment can be very challenging, both technically. And the secondary diffraction may occur because electron can easily be scattered. So the image from your. Sometimes can be. And also we just mentioned for transmission We need to cut the sample into very, very thin slices to make the electron to pass through the sample. So that is the treatment, and then the procedure itself is very difficult. And also the cost of Is pretty high. So let's make several examples using the technique we just mentioned above, we just mentioned before. To see how this technique, whether it's traditional or modern techniques, can help us to study certain biological questions. One is to study synapse, so let's look at what is a synapse? Synapse is where two neurons or two nerve cells, they connect to each other. And then transmits a signal from one to the others. And then, a little, we'll talk you about the details of synapse later. And now we'll just to see how this techniques can help us to to understand the synapse function better. So this is a synapse, this is a presynaptic terminal and then down here is postsynaptic terminal. And between that there's a space we call the synoptical cleft. And between presynaptic and postsynaptic. And in the presynaptic, normally we have a neurotransmitter. Is a certain type of chemical that can bind to the receptor at the postsynaptic membrane and it tells the postsynaptic site what to do. So this neurotransmitter often located inside of certain vesicles. And these vesicles can get down to the presynaptic membrane and then release the neurotransmitter. So the Can help us to see the detailed structure of synapse. This is a Picture for a synapse. And then this structure are mitochondria. Because of the synaptic release, the vesicle movement and release need lots of energy ATP. So mitochondria need to be there to produce ATP. This is the synapse studied by we use a super resolution image. If we want to study two synaptic proteins, one located in presynaptic side and the other one located to the postsynaptic side. Before, with the traditional confocal microscopy, if we labeled one protein as green the other one as red, we will see a block of yellow stain. Because we mentioned before the synapse collapse are above ten nanometers. So, all the things together, one pair of synapses is not bigger than 200 nanometers below our limits of optical diffraction. So we cannot see clearly the structure or the location of two proteins with the traditional. Confocal microscope. So if we use super resolution image that we can overcome or we can bypass this optical diffraction limits. That we can see clearly, the position of two sets of presynaptic and postsynaptic protein. So compared two masses, or two technique, this picture, the left one is the traditional confocal microscope. We don't see any clear precision here, it's just a whole blur. And then with the super resolution calculation that we can see a pair of green and red. Another pair of green and a red. And the precision of green and red are very clear. So if we blow up these pictures or these image. So the confocal, we only see pixels, we don't see any clear structure. And with the super resolution imaging, we can see clear blue ones and red ones, and blue ones and red ones. So with the help or with this technique, we can decide precisely where these protein presynaptic one and postsynaptic one located. And then she came into this schematic drawing of the locations or position of these synaptic protein. Say for example for presynaptic proteins, we have this and we have receptors located in this site, this synaptic cleft, the space in between. And then for postsynaptic protein we have NR2B, one subunit of NMDA receptor, we have GluR1, glutamate receptor one. And then we have PSD-95, Homer, the location, the position of this protein we can decide roughly the position of these both the pre and post synaptic proteins. Another example is vesicle movement. We mentioned before, the neurotransmitters are located inside of these vesicles and be carried by these vesicles. These vesicles can move towards the presynaptic membrane, get merged with, get fused and merged with presynaptic membrane. And then the stuff inside the neurotransmitter can be released from the vesicle into the cleft, into the region between pre and postsynaptic membrane. So there are several models to propose how these vesicles move and then get fused with presynaptic membrane. Model one is this fused and collapse model. It says the vesicles will move to the presynaptic membrane, this is a presynaptic membrane, this is the vesicle. And then vesicle membrane fuse with presynaptic membrane. They fuse together and then collapse. The vesicle itself collapse, and then inside neurotransmitter get released into the space between the cleft. There's another theory called kiss-and-run model. This model says the vesicle get moved close to the presynaptic membrane and will touch the membrane. And it will make a little hole there with the presynaptic membrane. And the neurotransmitter will get released through this hole. And then this hole will immediately get back to each other, will close the vesicle again. And then the vesicle can be recovered right after the neurotransmitter release. So, this process, this theory, this model here, the so-called kiss-and-run. So one kiss and then close again and run back propose a much higher turnover rate or recovery rate compare to the fuse-and-collapse model. There are physical evidence for fuse and the collapse model from the early years. But for kiss-and-run model, people propose it, but there's no very strong visible evidence to see the kiss-and-run model. Every evidence supporting the kiss-and-run model comes from the electron physiology recordings. Not the imaging study that we can really see this can happen in the life cell. So this paper, the last author is Richard Tsien. So he's a brother, older brother for Roger Tsien. So Roger Tsien made a very neat, very nice study to finally prove this kiss-and-run model and finally realized this kiss-and-run model in the cells. So the thing he does is, he used this, he take advantage of this so-called Qdot, quantum dots, the full name is quantum dots. The quantum dots is kind of a material, it's a man made material. The scale is nano, is under nano, it's in the nano scale. This particle is very small and you can make it theoretically into every fluorescence color, and then it has a very bright fluorescence. So, one single molecule can be observed in our just regular fluorescence microscope. So this particle is small, it can be in any color and it's bright, it's very bright. And it can be in any size because it's just a man-made material. So Roger, not Roger, so Richard take advantage of this particle. He made a Qdot, a quantum dots, with the size that can be uptake, can be endocytosis by the cell. And then, just cannot be exocytosis by the cell. So can be uptake by the cell, but can not be spilled out by the cell. So the vesicle can take this Qdot into the inner side of the vesicle, but it can not spill it out. So the trace, or the pathway of this quantum dots labels the movement or the pathway of this specific vesicle.
