Okay, and the observing can also be achieved into at a different molecule, different projection assembly and different synapse that we just discussed. Okay, for example, one can use an ingorged staining to look at the different layers of the cortex, okay? And that only allowed you to look at different layers. It does not achieve the molecular specificity. But again, with that, you already know in humans, we have six layers of the cortical region. And then for example, in layer four, can also be subdivided into different subregions, especially in the sensory cortex. What the sensory information actually project from the thalamus into the layer four. And then clear process by the different cortical region and then get output in the layer five actually. And different part of the cortex might have different layers. And the image message is very powerful because it can allow for non-invasive and parallel information processing. And there are different ways to achieve the imaging. For example, confocal, you introduce a pin hole, will allow it to only acquire the imaging from the focal plane, while rejecting the lights getting from the other focal plane. So this will achieve a much higher resolution, especially in the sick tissues, okay? And people actually have invent newer methods, for example, this is the light sheet microscope. So rather than deliver illumination light from a path, if you focus a light from the sideways, okay, and this light will be focused by this lens from the side, okay? And it will illuminate, it will only activate a sheet of the cell, which can achieve much higher C resolution, comparing with the commission method. So the light sheet microscope is being very powerful to have a fast acquisition speed and that's phototoxicity, because you only illuminate much less number of cells. And there are also masses trying to make the non-transparent brain transparent, okay. The reason is that we are interested about mouse, human brain. And they are not transparent, okay. And zebra fish are transparent. But people have been developing different techniques to clear the brain, okay. So for example, this will illustrate one of the ways called clarity, that you can use some special combination of clearing solution, okay, for example, some special refreshing index of solution. And some of these clearing techniques to eliminate lipids from the cell will maintain the brain's anatomy structure, and for example, in this case, there's a technique, so-called clarity. One can make the mouse brain transparent and then one can readily image him, many GIP labelled transgenic mice, okay. And so far this technique can only be used in a dead brain that has been fixed, okay, because all this clearing technique requires the brain to be fixed to maintain a structure. Okay, and a lot of them has a huge change of the brain's fluid. So, all the cells are dead. But the reason is that the brain are so complicated, even for the fixed brain. We still have not achieved the neuronal connecting connectomics. So people have to achieve even in the fixed brain their precise connections, mapping. Then one can hope in the dynamic brain to understand all of their temporal dynamics. So this carotid technique or other cleaning technique is another technique skill are also very useful. And again, Ramonica Hall, 100 years ago, already using a gorgeous staining to allow one to see single cell. And then you can look at how this cell project into different part of the brain. For example, this illustrates a cell in the olfactory passageway that can project in a different part of the brain, including the olfactory tubercle and cortical amygdala in different brain region that are supposed to involve in different information processing. For example, amygdala is related to the fear conditioning, that how a mouse or human sense fear, and mouse, once it sense fear, it will immobilize, okay, it will not move. Okay, and this illustrate how this olfactory information gets projected into this neuron and this neuron sending out information into a different part of the brain. And one has to develop combining with the genetic techniques and the single cell labelling. One can look at a single cell labelled, for example, with the TAP, and you can easily see this is a sub cell of the structure, for example, this ventricular spine. So this opens up new ways to look at a live animal, because of the gorgeous staining a single cell labelling, again, dealing with a dead brain, okay? For the GIP, one can look at it in a live cell, and if one can pull a GIP instead of just putting purely for instance, you can put in some genetic sensor that can sense the neuronal activity. One can image that neuron's activity in the live animal during TAP. And again, there is a single cell labelling technique that I just mentioned that induce recombination. One can achieve the first labelling and to the homozygous mutant in one cell, and this can allow one to do linear tracing. For example, one can use the flippase expression to induce this recombination, and these recombined cells will be marked green and then you can see only a single cell. How do you achieve this flippase express only in this cell? Well, we already discussed this flippase can be controlled by a promoter. And if this promoter you are using are tissue specific, you will only get expressed in this lineage, okay. But how do you control it expressed only at this top? Well, we already talk about, we can use some chemical method. For example, the petrol cycline or dorsal cycline induced transcription factor to express by giving the chemicals to the cell or to the fly or to the animal. On the other hand, in the fly, people can also use the heat inducing promoter, heat shock promoter, so at a certain time, you can just structure the fly into heat shock for very brief period of time. There is some special promoter that will be heat sensitive that will only return at that time. And then you can induce by a temporary specimen, okay? And this is an example of illustrating a fly neuron and see how elaborate their neuronal neurites might be. And this is a specific ways. And electron microscopy, because of its super good spatial resolution, it can allow one to look at which molecule at what time to work. For example, this illustrates immunal code labelling of one specific molecule called dynamin. And we talk about it briefly in our exo and endocytosis. And this illustrate actually a synaptic vesicle, once it gets exocytosis and once it needs to be regenerated, it actually require a GTPase called dynamin. And this dynamin if you do the immunal code labelling, interesting you'll find, you only decorate in the ring of this internalized vesicle in the neck, okay, forming a ring. Okay, so this gives people clues of how this dynamic work. So it work as a seesaw that sort of pinching off this vesicle, and once this vesicle get pinching off, they can refuel with the transmitters. And so dynamin is working in internalization step using the GTPase activity to fission the vesicles. And the most recent development of this is super resolution imaging, for example, this illustrates that there's a presynaptic marker for some and a postsynaptic marker homo-1, okay? If you are using the conventional confocal or other light microscopy techniques, what you observe is the green and the red acololytes, because they are actually labelling a synapse, the presynaptic and postsynaptic cells. Because they are so close to each other, the commissioner light microscope cannot read softly. And then if you're using the super resolution imaging, and then you can clearly see they are person in homo-1. They are separate with certain distance. And then you can do that with alumonical in your phone actually. The presynaptic and postsynaptic are well organized structure and different molecule, sent to a light at a different distance to each other. For example, the piccolo is a little bit far away than the receptors are closer to the synaptic cleft, okay. And likewise, the glutamate receptor are the postsynaptic receptor that will sense the transmitters, and they are the organizing molecule, the scaffolding molecule, PSD-95 or homo-1. In the PST are a little bit far away from the membrane, including the glutamate receptors. And again, these are difficult to achieve with just the electron microscopy because the electron microscopy is very difficult to get all those molecular identity. A lot of times, you only can get one immunogoal to work. And probably most of the people cannot get two immunogoal to work at the same time, not to mention with so many different molecules, okay. And this illustrates the enlarged, you can see at every synapse depends on their orientation. You can clearly see the segregation of this pre and post synapse of terminals. And there are other techniques that people can image in the information flow across different brain regions. And this is at a more global level, okay. And for example, one could inject virus locally and then looking at its projection, okay. So for example, if you inject virus here and then this virus will infect the cell. For example, if the virus only infect the cell in injection region, okay, then you can see how these cells get projected to the other region. Because you infected the virus, so the virus are labeled with some GIP molecule for example. And this GIP molecule can express in those infected neurons, and then they will fill the whole neuron. And therefore, you can see, how these cells project to this region. And what you can do is then if you inject the virus with a different color, or the same virus in different region, in different brain, and then you can establish roughly how this projection might work, might organize, okay. This requires that a virus only infect the cell in the injecting region, okay. If the virus infect this cell and this virus reproduce and then gets secreted here, and then, this cell will also get infected, they will inject somewhere else. And then the other cell will also get secreted and then they will inject somewhere else, and then what you are going to see is that the whole brain will be lighting up. And then you are losing the projection specificity. How you can do that to limit the spread of the virus? Well, you can, for example, because virus replicate takes time, right? So you can limit the time to imaging, only allow them to project and not to reproduce, okay? But people actually have invent methods. So this is illustrated not just using one color, but you can use multiple color to label different cells. This is in flies using a technique similar as a brain bulb, and you can look at the different cells are labeled, but different combination of flows and protein. Okay, you can see how one cell label the entire eyes of a fly's brain. Okay, this is a fly's eye. And one advantage of this labelling is if you are taking good, colorful images, this provides good screen saver for your desktop, okay, and then you can submit this beautiful imaging to the contest. For example, recently, the School of Life Sciences has this imaging contest and then you can win some prize by submitting your images, okay.
