My name is Johanna Hoog and I am an assistant professor at Department of Chemistry and Molecular Biology. At the University of Gothenburg. Today, I am going to tell you about the electron microscopy of extracellular vesicles. And this lecture will be in three parts, of which part one will be an introduction. To all the Techniques that have been used to study extracellular vesicles. The first thing I would like to focus on is, that electron microscopy is not just one technique but a wealth of many different techniques. And you need to know which technique to use when, to be able to address your scientific question. The first separation we can do is, between scanning electron microscopy and transmission electron microscopy. In scanning electron microscopy, cells or organisms are covered with metals. And the electrons from the scanning electron microscopes are scattered on the surface of the sample. And this is what makes the image of the samples surface. In transmission electron microscopy, you have to have very thin sections or thin samples. To be able to send the electrons through the sample and then, you can image also what is internally inside. For example, the nucleus or the smooth As we have here. Scanning electron microscopy hasn't been used very extensively in extra cellular vehicles research. Scanning electron microscopy has a few different techniques associated with it. The Conventional one is the one I just showed you an example of, but there is also other techniques which makes large 3D reconstructions. By using a micro tool or a focus IM/b beam inside of the scanning microscope that slices off a section. And then, you can image the next section. You slice off a section and you image the next section and like that you can reconstruct large volumes of 3D tissues or cells. So, so far, I think only conventional SEM has been used to illustrate extracellular vesicles. Which is quite logical because they're small structures. Here we see an example of extracellular vesicles on the outside of cyanobacteria. And so, as you can see, you can see the sizes of the vesicle and their association with the cells at the surface. Transmission electron microscopy actually offers a wealth of different techniques. And we are now going to focus on the ones that have been used to start the extra-cellular vesicles. And I think the one that you're most familiar with, and every person in the extra-cellular vesicles field are very familiar with. Is negatively stained vesicles, so negative stain is a very simple technique. Where the sample preparation can be limited to less than half an hour. And what you do is, you take a mixture of vesicles on top of an electromicroscopic grid. And you let them sit there for five to ten minutes and hope that they adhere to the Grid. Then you might add affixative, although the need for affixative in this step is not yet proven. Because it's not used in other fields when you do negative stain. But the fixative might improve the retention of vesicles unto the Grid or possibly also the EV structure. This needs to be elucidated after the fixation and the following washes. You add the heavy metal and let it dehydrate and the dehydration then forms a shell or a coat of metal on top of the vesicles. Which will then reflect the shape of the vesicles beneath it. Here's an example of a negatively stained image that was published by Madal. And as you can see, 0the vesicles quite often look like doughnut shapes. This doughnut shape is an artifact of possibly sample dehydration as a process of the sample preparation. Now we're going to move on a more traditional Technique that is used often on cells. We can also be used to good effect in the exocellular vesicle sample and that is thin section electron microscopy. And we will start here or focus here, on chemically fixed samples, because that's what people have published so far. So when you want to chemically fix extracellular vesicles, you will start with the isolated vesicles. You will add the gluteraldehyde or other chemical fixative. Then, your sample will be embedded into a plastic and this plastic stub is then put into the microtome. And in the microtome, you can do very thin sections of this sample. Usually 40-80 nanometer thick and you put them on to the Grid. This is then floated on heavy metals to further stain the sections and provide contrast in the electro microscope. Here's a beautiful example of what you can see, what thin section electron microscopy. And this is actually from one of the first publications of extracellular vesicles. That were isolated from human seminal fluid and there called prostasomes. You can see that there's a full of something electron dance in the lumen of the extracellular vesicles. But you can also see a very clear bilayer and if we look at the other examples here. We can see several bilayer on this vesicle and over here, we can see a intricate vesicle with the smaller one inside of larger vesicle. So these kind of details you can easily see with thin section electron microscopy. Now, we will move over Cryo-electron microscopy. And this is when the microscope is chilled with liquid nitrogen and you're imaging the samples in ice. So, we will first look at conventional Cryo-EM and here are the sample preparations is relatively simple. Where you have the isolated vesicles and you put them straight onto the Grid. And then, you block it with a filter paper and then plunge freeze it immediately into liquid ethane or propane bath or mixture of the two. And in here, they will freeze so quickly that the crystals will not have time to form. And hopefully, you'll have an amorphous ice to display In a very beautiful manner. So, here is an example where you can see the lipid bilayer of the vesicle inside of the eyes. What we have to focus on here is, that there is no fixatives involved and no heavy metals involved. And that's also why the contrast, the difference between the gray levels here. Are not so high as you would see in a chemically fixed sample for example. You can also look at the cryo-electron microscopy and do a tomography with it and this is quite an advanced technique. But can be very helpful for a certain questions in the EV research field. So, how do you do electron tomography? Well, if you put an object in the electron microscope, you will tilt the object and take pictures of it. 2D pictures and many different angles and as you take these pictures, you create the tilt series. This tilt series is then perfectly aligned to each other. And then, you can create the 3D volume you originally imaged back in the computer. So, you now have reconstructed the 3D volume of what you originally looked at. Cryo-electron tomography has been preformed several times on extra cellular vesicles. Here actually, I think are all the published electron tomography to date that has been done on EV's. And the benefit of electron tomography is that you can create slices. Electronic slices through this reconstructed three D volume. And these slices are as big the pixel size when you acquire the image. So, here's a one nanometer thick sliced through an extracellular vesicle and as you can see, there is a vesicle inside of that vesicle. This amount of detail, you could not get from conventional Cryo-EM. Here now, is a summary of all the techniques that I have shown you that has been used in Extracellure Vessicle research. And I would like to point out particularly these two techniques here high pressure freezing and thin-section electron microscopy. And correlative light and electron microscopy is two areas where EV field can definitely advance. Because they haven't been used here yet and I think high pressure freezing is definitely to be advised above chemical fixation. As it is a structural improvement of the sample and the correlative light-electron microscopy. I think, will be very useful for the future study of extracellular vesicles to release and maybe able so, absorption of target cells. So now I would like to thank you very much for listening to Part one. And I hope you will come back and listen to Part two and three of this lecture on Of extra-cellular vessicles. Thank you very much for your attention, and here are the copyright licenses for all the Papers that we've used in this lecture.
