Vesicles were introduced the technique of cryo-transmission electron microscopy and we'll present its application to the study of extracellular vesicles or simplicity I will say priority for cryo-transmission electron microscopy and EVs for extracellular vesicle. Here is the outline of the course with two main parts. Part I, imaging EVs by cryo-TEM. Part II, phenotyping EVs by cryo-TEM combined with Immuno-gold-labeling. First, what is the principle of Cryo-TEM? A small drop of a liquid sample is deposited on an electron microscopy grid. The grid is quickly plunged in a cryogen. Then it is transferred in a cryo-electron microscope. Where it is observed [INAUDIBLE]. What occurs is that upon quick freezing, a thin liquid film of water, buffer, body fluid, is transformed into an amorphous solid. The objects contained in this film are observed in their native frozen hydrated state. In practice, on the left is presented a scheme of a plunger used for freezing thin liquid films. A four microliter droplet of a solution say an EV containing solution is deposited on an electron microscope figurine. Then with a piece of little paper, most of the liquid is removed and the grid is plunged quickly in a cryogen using This guillotine type of machine. Liquid ethane cooled by liquid nitrogen is the preferred cryogen system. After this, liquid is transferred into liquid nitrogen. It's mounted in acquired order and inserted in the electron microscope. And here on the right, we're within the electron microscope. The grid and the metal droplets of frozen material will maintain a cold temperature, about -170 degrees Celsius. What is critical is that after freezing, samples must always be kept at cold temperature. At no stage in which the water or put in air otherwise, this thin liquid film will evaporate and the specimen will be destroyed. The maximum film thickness is usually about 300 to 500 nanometers. And further on the Observations must be performed at low electron doses In order to avoid irradiation or being damaged. Let's look at some images of EVs by cryo-TEM. These are typical images of EVs in pure and processed blood plasma. As you can see here EVs appear as circular objects. And this is the case for the vast majority of EVs in all body fluids analyzed. The size can be measured precisely. This one on the left has a diameter of 180 nanometers the two ones here on the right diameters of 65 and 45 nanometers respectively. As you can see the meteor shown in these looks green. This is simply due to the presence of a large amount of proteins in plasma about 16 milligram or milliliter. In cryo-TEM, it is appear surrounded by two thin dark lines. Which correspond to the two leaflets of flipping membranes or come back with the point in a moment. In conclusion, what we have observe here are EV's embedded in a thin film of frozen plasma The white asterisks you see here and here point to the theorem. In fact, it is a. The nano droplets of frozen plasma are suspended within the holes of the What are the advantages and limitations of cryo TEM. First, general advantages cryo-TEM is the least invasive Technique. It involves no drying, no staining, no chemical fixation, no thin-sectioning. Objects are observed in their near native state, and their size and morphology are preserved. Basically what is present in the test tube in a solid state are not in a liquid state. The resolution of cryo-TEM images is [INAUDIBLE] say about 2 nanometers Or even better after image analysis. The contrast of cryo-TEM images reflects the local composition of the specimen. And there some of advantages of cryo-TEM in EVs analysis. If it just so, it is possible to image EVs in pure body fluids. And running of presence of I putting concentrations. Therefore, there is no need for sample processing like centrifugation or filtration of precipitation Filtration which may cause artifacts. Like EV aggregation the lipid bilayer structure is well resolved as we saw previously, and this allows distinguishing Evs from other type of contaminating particles. Let's come back to this point by the way, in cryo-TEM images. A lipid bilayer appears as two thin dark lines separated by about 4 nm, which is the expected width for a lipid membrane. On the left here is a synthetic liposome in buffer. On the right is a native EV in blood plasma. This contrast is simply due to the fact that phosphorous atoms, which are the heaviest atoms of lipid molecules which are represented with these blue dots. These are the phosphorous atoms. Phosphorous atoms are located symmetrically at the opposite extremities of lipid molecules. And being heavier than the other atoms, they scatter more electrons, which explains these two dark lines. Now what are the main limitations of cryo-TEM? There is a need for expensive equipment but even more important there is a need for specific training and expertise. Frozen-hydrated specimens are extremely sensitive to electron beam irradiation yet this is easily solved by using low electron doses. On cryo-TEM is in general is a slow technique. Only few samples, say two to ten depending on the nature of the sample on the question which is asked can be analyzed per day. Some of the examples of EVs image by cryo-TEM On the left [INAUDIBLE] EVs are observed next to this sperm tale. On the large elongated EV [INAUDIBLE]. This corresponds to membrane [INAUDIBLE] distributed homogeneously. On the right, EV's in [INAUDIBLE]. Note the prisms of [INAUDIBLE] and straight tibula EV's labeled T next to [INAUDIBLE] labeled s. Let's move to the second part of this course. Phenotyping EV's by Cryo-TEM combined with immuno-gold-labeling and I will set immuno-Cryo-TEM. The principle of immuno-Cryo-TEM, gold particles are conjugated with a ligand a protein an antibody and are used to label receptor molecules exposed at EV surface this allows identifying their cell origin Or phenotype. Gold particles present high contrast in Thanks to their large electron scattering property. Particles of two nanometer diameter or larger are easily detected by. Schematically, Protein-conjugated gold particles, are used as flags to identify EVs' origin. Some examples of EV phenotyping. These images are taken from a study of EVs exposing phosphatidyserine [INAUDIBLE] in blood plasma. Indeed, it is exposing PS are considered to be a major population according to classical theory of [INAUDIBLE]. In order to identify PS exposing Annexin-5 conjugated gold particles are used because Annexin-5 is a of this. On the left, you see an EV densely covered with black dots. Each black dot is a gold particle of four nanometer diameter conjugated with Annexin-5. This image demonstrates therefore that this EV exposes [INAUDIBLE]. On the right you see 2 EVs, the small one which is densely labeled Annexin-5 gold particles [INAUDIBLE] of 10 millimeter diameter. While this one is entirely divide of gold particles. Therefore, this EV, the bottom one is PS positive and the top one is PS negative. Two of the examples of two EV phenotyping by immuno-cryo-TEM. On the left, a long tibula EV densely [INAUDIBLE] with gold particles conjugated with an antibody directed against CD41. And CD41 is a showcase marker of blood platelets. Therefore this area is of platelet origin. On the right, the result of a double labeling experiment performed on a plasma cell Central from a drepano disease patient. The large gold particles are conjugated with antibody directly against CD dues refi or glyco foreign which is a mark out of high blood cells. While the small particles are falling in the diameter, are conjugated to annex [INAUDIBLE]. The presence of both types of gold particles on this [INAUDIBLE] indicates that it is of red blood cell origin and in addition, that it exposes peers on its surface. Here again, note the well resolved Lipid bilayer structure. What are the advantages and limitations of immuno-cryo-TEM? Advantages, immuno-cryo-TEM provides a unique way to identify the cell origin of EVs. Single-step labeling avoids washing and centrifugation steps which are lengthy and may induce specimen modifications. Labeling is highly specific this was clear from the images we just saw. The limitations, there are basically no limitations. There is a constraint that the synthesis of protein-conjugated gold particles requires expertise in protein chemistry. Many thanks for your attention.
