We're now going to talk about the second part of the electron microscopy of extracellular vesicles. And now, we are going to focus on pros and cons of using each particular technique that I introduced in Part 1. The first technique that we will discuss is the most commonly used technique which is negative stain of transmission electron microscopy of negatively stained samples. Negative stain is a very variable technique as you can see here, the three different examples, the contrast between samples can change a lot and usually depends on how the samples are being prepared. But often there's also huge differences with one single user staining different Grids in exactly the same way and they can come out differently. What this really depends on, we don't quite know. But things such as sample concentration might have an impact of the final outcome. Negative stain is often used because is the fastest of all Techniques. It takes about 25 minutes, 20 to 25 minutes to prepare samples, and it confirms EV presence easily. Of course, it can't completely confirm the EV presence because with negative stain, you cannot see the lipid bilayer. So, it's a way to see that there's particles, round particles in your preparation, but note that they are EVs, if you should be really critical. The negative part also is that there's extremely variable results and the extracellular vesicles that you see have untrue morphology due to the chemical fixation in the hydration in the sample preparation. Also, quantification is not quite doable using negative stain. So for example, if you wanted to measure the diameters of the vesicles that you are seeing. We have done a test, and we show that the selection of what we call an EV in negative stain is very individual. And in that case, you would have to do a comparative study using only the same person to quantify all the images that you have. Also, the method might select for certain EV subcategories. For example, you have might pre-prepared your grids with different ways of charging the grid with UV light or with glow discharging. And this might select for a certain subpopulation of EVs more than another. So the next method that we're going to discuss the pros and cons on is the thin section on chemical fixation and high pressure freezing. So, here's an example of a multivesicular body that I've taken myself. It's from a high pressure frozen cell and you can see the vesicles and their bilayers inside of a larger vesicle. So this is very nice because you can see the bilayers nice and clearly. However, you can't really tell that this is a round vesicle from what you see there because you have only got a thin section of the vesicle and you might be looking at the round structure. Or you might be looking at the very elliptical structure, like a tubular structure, and you would never know by one single thin section. So both of them will look round and have a bilayer in a small cross section. And what about, if you looked at cell that looks like this where they have a very hairy surface? All these hairs on the outside or the extensions of the cells on the outside, they might be looking like extracellular vesicle in a cross section. And it's therefore very important to be critical about what you call an extracellular vesicle when you look at them in the social connection with the cell. There is further limitation with using thin section. And that is for example, that if you catch this section up here, quite high up to the top of the vesicle, you will get quite a slim diameter. But if you have the same vesicle, in the middle of the vesicle, you will have a larger diameter. And of course again, at the bottom you will have slimmer diameter. So to be able to reconstruct or measure the diameter vesicle using thin sections, you have to do a serial section reconstruction. And to do that for each vesicle that you see seems to be an awful lot of work and I would recommend to use another technique to measure the diameters of the vesicles that would be easier. So the pros of using thin section electron microscopy is the bilayers are visible so you can definitely say that this is membrane enclosed organelle. And you can say that larger EVs can also be visualized, because for example, methods like a cryo-electron microscopy, you cannot visualize anything larger than half a micron. But in thin sections you could visualize as big things as you want. You also have very good contrast because you have been able to add heavy metals in the sample preparation protocol. But the cons is that you have a slow sample preparation and it's normal to take between three, four days to a week to prepare samples for thin section electron microscopy. And you, of course, have to be trained in doing so. It cannot be used to measure the EV diameter and of course, the samples are fixed with some sort of maybe fixatives like glutaraldehyde or high pressure freezing. But then heavy metals are introduced in any stages on both thin sections verticals. So here, I have prepared a question for you to show you what is difficult when you want to see extracellular vesicles when they are prepared in close connectivity to a cell. So do you think that A or B or C are the extracellular vesicles on this picture? I will give you a second to think about that. So actually, I would say that C are the extracellular vesicles and the reason why I say so is because they have a very different texture from the cytoplasm. And we know that the extracellular vesicles don't contain ribosomes. And also, you can see here this extension from the cells so we know that these are cells with extensions on the surface. And I think that these things that are very similar in the texture from the cytoplasm are actually just extensions seen coming from above or below, and they appear as an extracellular vesicle. And the same is with A. But in C, you see that they're very different in the texture. You can also see that they are, well, if you zoomed in you could see that they are lipid bilayers. So what is the pros and cons with using cryo-EM to look at extracellular vesicles? While using cryo-EM was first shown that these EVs are actually not donut shaped as was originally thought by looking at negative stain. And here, you can see a nice arrangement of images using cryo-electon microscopy. And what I would like to point out, particularly here, is a nice example from Arraud et al., where they show the bilayer of the vesicle. So this is very much a way to show that this is an extracellular vesicle, and not a particle that we are looking at. But there is limitations also to cryo-electron microscopy because you send the electrons through the entire thickness of the ice, and as you're taking that picture, you don't know if these two vesicles are above each other, like it is here. You see a picture that will look like this, with a larger vesicle and a smaller vesicle in the same area. But they could be equally be a larger vesicle containing a small vesicle and the picture will look the same because what you're looking at is a 2D projection of the whole ice volume. So the pros with using cryo-electron microscopy is that you have no fixatives or heavy metals in this sample preparation method. And that's of course a very heavy aspect of better sample preparations. And also, the bilayers of the membrane can be visualized and that is very nice for EV researchers. The sample preparation is relatively fast if you have been trained at it. And you can get about 1 nanometer resolution in x and y. And of course, instead you will have the resolution of the thickness of your ice which is usually between 100 nanometers and 500 nanometers. If it's thicker than that, you will not be able to see anything. So it requires more specialist equipment and training, so the electron microscope that you use has to be suitable for cryo-electron microscopy, and you have to be trained to do this, because it's hard not to get ice contaminations on your sample. And of course, you cannot visualize anything thicker than half micrometer. And that is probably the biggest caveat with cryo-electron microscopy because very few things are smaller than half a micron in the biological world. We are just very lucky as EV researchers that most the EV populations are smaller than half a micron. We have very little contrast in cryo-electron microscopy and that is because no heavy metals was used in sample preparation. And of course, that's the whole benefit of using cryo-EM so that's just something we have to live with. The Z-resolution is the thickness of the ice and so we can think that vesicles are within each other without having proof of that because they are actually maybe two vesicles laying above and below each other in the ice volume. Also, the sample preparation method might be selective because there might be different affinities to the extracellular vesicles to the grid. And also, you might be blotting because you have to blot away excess liquid when you prepare EVs for cryo-EM. And there might be selective blotting, maybe larger or smaller vesicles are blotted off faster than the other. And therefore, your sample might be a little bit skewed. I'm now going to talk about electron tomography, so cryo-electron tomography. And I have already introduced it to you in Part 1, but of course the best benefit of cryo-electron tomography is that you reconstruct the entire 3D volume of the ice that you're interested in. And you can, as such, definitely see the difference between two vesicles inside of each other other or vesicles above and below each other. So with electron tomography, you can definitely prove if you have vesicles inside of other vesicles. So here we have an example where you take your picture here in the x and y plane and you will see a nice vesicle. But then, since you have reconstructed 3D volume, you can also take a picture in the x -z plane. And here, you can see that you have a larger vesicle and a smaller vesicle, and it's very clear case. However, even electron tomography, it has its drawbacks. And one of them is that we cannot take pictures of the sample from all different angles, so we usually go +-60 to 70 degrees when we take this tilt series. But at some point, you start seeing the holder that is holding the sample that you're looking at. And, therefore, you can't go +-90 degrees, which of course would yield the best reconstructions. So there is a part of this area, the vesicle, that haven't been imaged. And that creates an artifact in our electron thermograms, which you should call the missing wedge. So this is the missing wedge here. And of course, when you look at the vesicle, you can see it very clearly in the x and y plane. And here, you can see a vesicle within a vesicle. But you can see here on my reconstruction that I have a very flat surface on top of the vesicle and that is because I've just added a cap because I assumed that there is a membrane there. But I haven't actually seen that membrane, so that's one of doing it using prior knowledge that this should be complete EV. But it's of course, my interpretation of this vesicle. In the Coleman et al., paper of 2012, I think they were the first people to do electron tomography of extracellular vesicles. They chose to present their data in a different way, where they have shown the openness of their model and actually use that to display what they found inside of the model which is another vesicle. So actually, you can choose to display your electron tomography the way you want. And the main point here is maybe to think that the colorful image, the 3D image that we show, is the author's interpretation of the data and the real data is the black and white picture. So, the pros and cons with the cryo-electron tomography is that you will gain 1 nanometer resolution in all directions, if you want to, of course it depends on the pixel size that you use for acquiring your tilt series. And you can get 3D models of EVs and therefore, you can with decisiveness say if a vesicle inside of another vesicle. But it's much more time consuming to acquire the images. Usually tilt series will take something between 25 minutes and an hour. But then the reconstruction and analysis is much more time consuming than that. And it also requires knowledge of specialist software and that's something that's very few people are, well, not so many people are experts at. And then, of course, you have the missing wedge artifact which makes the vesicles appear incomplete in the 3D volume of the reconstruction. So, thank you very much for your attention and I hope you will come back to listen to how you can find individual proteins inside of electron microscopical sample preparations. Thank you.