Now a fundamental limitation of traditional thin-section EM and staining and shadowing techniques is that they involve dehydrating the sample. And of course living cells are hydrated. So there's no guarantee that the molecules inside will maintain their structure through the dehydration process. The next two techniques involve freezing cells in their hydrated environments. The first one we'll talk about is high pressure freezing. In the first step, a block of tissue is placed in a high pressure freezer. And this device starts by applying tremendous pressure on the block of tissue and then rapidly cooling it by applying liquid nitrogen all around it. The purpose of the pressure is to decelerate ice crystal formation. Now we know that if you take liquid water and you slowly cool it, the water molecules will bounce around until they find hydrogen bonding partners to form an extended ice crystal. And the ice crystal is less dense than the liquid water. And so the, the water expands as it crystallizes and this of course would destroy the cell. However, if you apply very high pressure to a block of tissue so that it can't expand and then you cool it, the water can't expand in an ice crystal. And it stays more nearly in a liquid like state. There still may be lots of little micro crystals but they don't grow and rip the cells apart as they would if you very slowly cooled the cells at atmospheric pressure. And so the pressure prevents ice crystal formation. In the next step, the freeze substitution while the block of tissue is still held below freezing temperature, it can be dehydrated and infiltrated with an organic solvent and finally with a, a plastic resin that will polymerize, even at below freezing temperatures. And this is what's called low-temperature embedding. And so, starting with a high-pressure, frozen block of material, it can be plastic embedded all before it really thaws completely. And this preserves the native structure of the cells. Once it's plastic embedded it can be warmed to room temperature, sectioned as before, typically stained and then it can be imaged in the electron microscope. Now of course, if it's plastic embedded and sectioned, and stained with metals, again, the actual biological macromolecules will not be seen, it will be the stains that delineate them. But nevertheless by high pressure freezing, free substituting, and low temperature embedding, the basic structure of the cell is better preserved. These two images show an, an extreme example of how high pressure freezing and low temperature embedding can better preserve cellular structure than chemical fixation alone. So, these are images of Multivesicular bodies, courtesy of Mark Ladinsky, and so the image on the right here is of a Multivesicular body that was preserved with Ultra-Rapid Freezing and Freeze-Substitution, or high pressure freezing and low temperature embedding free substitution. So what you see is a series of membranes concentric vesicles. Each one you can see the two leaflets of the bio layer. So this is one membrane forming a vesicle. And inside is another vesicle with its two leaflets of its membrane here inside. And they're just concentrically arranged. Probably 15 vesicles all within each other here in this Multivesicular body. And they're next to two other membranes of the nuclear compartment and these two membranes you can also see the two leaflets of the bilayer. So these are nicely preserved, the membranes are smooth and clear. In, in a different example of a Multivesicular body shown on the left, this is a tissue that was fixed by traditional chemical fixation at room temperature. And then it was plastic embedded, etc. And in this case, you can see that there were a lot of membranes on top of each other, stacked membranes. But the membranes are wavy there seems to be large gaps in between. Sometimes in traditional chemical fixation, there's material lost. It's extracted away. And so clearly, in cases like these, the high pressure freezing, better preserves the native structure. Now of course it would be better, if we could take the frozen material and image it directly in the electron microscope. But we run into another fundamental problem. Which is that the imaging electrons are so strongly scattered by biological materials, that the mean free path link of, for instance, a 300 kilovolt electron in a cell is only about 260 nanometers. Meaning that your average electron with 300 kilovolts of energy only passes through 260 nanometers of material before it's scattered. And if it's scattered multiple times or if it's inelastically scattered then it doesn't contribute usefully to the image anymore. And because of this, there's a fundamental limit that we cannot image samples that are thicker than about a half a micron. So in any sample preparation technique, it has to end with a sample that's about 500 nanometers thick or less. Nevertheless, if we want to image a frozen cell within the electron microscope, it is possible to first high pressure freeze a block of tissue and then section it in the frozen state, a process that we call cryo-sectioning. This is a movie that was recorded by my colleague, Alistair McDowell and his colleagues and what we see is a high pressure frozen block of tissue at the end of a microtome arm. And this here is a diamond knife. And as the microtome arm is brought back and forth across the sharp edge of the diamond knife, very thin sections of that high pressure frozen material are being cut off. And they stick to one another so that they emerge as a ribbon of sections. And then down here we see an eyelash that's being used to catch that ribbon of sections and draw it forward. And so it is possible to cryo-section high pressure frozen material and take that ribbon of cryo-sections and then lay them down on an EM grid. And when that ribbon of cryo-sections is laid across an EM grid, it, it can look like this. This is an SEM image of a ribbon of cryo-sections laid down across an electron microscope grid. Now, one of the challenges of this technique is that the cryo-sections themselves are often crumpled. And crinkled. And, so, here we see in the SEM image of the, the cryosection laying on the grid that the section isn't even nearly flat. And there's also a lot of contamination on this particular cryosection. So, one of the challenges is to get the cryosection to lay down flat on the EM grid. Sometimes people will take a blunt instrument, cooled by liquid nitrogen, and simply try to press and crush the section down on to the EM grid, but it's only partially successful. In addition, when such cryosections have been imaged, it's been observed that there's a lot of compression that occurs during the sectioning process. So these are images of bacterial cells that were high-pressure frozen, cryosectioned, and then imaged on an EM grid. There are rod-shaped bacteria, and so they normally look cylindrical, but in this case, after they're cryosectioned, they're compressed by 20 or 30% in the direction of the cutting blade on the diamond knife, and so they're compressed, and this introduces artifacts into the image. In addition to being compressed, as the high pressure frozen material scrapes across the diamond knife, there are knife marks. And the knife marks in these images are represented by the black arrows where you see streaks that are streaking down across the image. Those are from imperfections in the diamond knife. A final problem arises because of the geometry of the cutting situation. Imagine that this is the diamond knife and the material to be cut is above that knife mounted into the chuck of the microtome. And this microtome arm is swinging up and down and up and down, in front of the diamond knife. And so this is going to be swinging down against the diamond knife. And as it swings down into say, this position, you can see that it will be cut by the diamond knife. However, the geometry of the situation creates stresses right here at the junction. And these stresses force material to erupt up out of the surface, creating a series of ridges, and crevasses on the side of the material away from the diamond knife blade. So cryosections suffer from crevassing on the surface opposite of the diamond knife. They suffer from compression as they slide into the blade. It, it compresses the material in this direction. And finally, there are knife marks that arise as the knife scrapes through and cuts the frozen block. So you see the shadows that appear going cross the section. These are crevasses that were caused by the outside surface being crushed on top of itself. Nevertheless this allows us to obtain images of frozen material without chemical fixatives or stains or any classic embedding. The last sample preparation technique that we'll talk about is plunge freezing. In plunge freezing, a very small amount of sample is placed on an EM grid, suspended above a reservoir of liquid ethane, typically. And the liquid ethane is cooled by being surrounded by liquid nitrogen. So, the ethane is at approximately 80 Kelvin and then the sample is rapidly plunged into the liquid ethane. And if the sample is thin enough, heat comes out of the sample so quickly, and into the ethane, that the water molecules don't have time to bounce around and find partners to hydrogen bond with so that they can't form an extended crystal. Instead, the kinetic energy comes out so quickly that the water molecules and all the other molecules in present, simply stop moving wherever they are before they have a chance to crystallize. And all molecular rearrangements are frozen. Now I'll show a movie that we recorded for the journal of visualized experiments, showing this process of plunge-freezing. First, individual EM grids are loaded into an automatic freezing device. And, so, here the sample is being applied, a couple microliters of the sample is applied to the EM grid, and then it's lifted up in between two pads of filter paper. Those pads press against the grid, absorbing most of the material, and then the grid is plunged into a reservoir of liquid ethane. There it is in the middle. And the ethane is surrounded by liquid nitrogen. Once it's plunged frozen, it needs to be held in that frozen state. So this is an example image of two bacterial cells that have been plunged frozen across an EM grid. In this case we used the EM grid type called Quantifoils that have circular holes in the carbon film surrounding them. So that, that's a circular hole in the grid surface itself. The rest of it, this is a carbon film. And we simply took a growing bacterial culture, and spread it into a very thin film across that grid, and then plunged it into liquid ethane. And then by keeping it frozen constantly, from the time it was plunged frozen until it's in the column of the electron microscope, we can then take an image through it, and so here is what you see. The great advantage is that we've preserved the cells in a near native state. In fact, it's been shown that if you take a grid like this and thaw it out in growth media, many of the cells will recover life and they'll begin to replicate. And so we're looking here at a cell in a native state and all the molecules are where they were when we dropped it into the liquid ethane. There's no chemical fixatives altering the structure, there was no dehydration because they're still surrounded by the water, there is no plastic embedding, there's no sectioning or staining. It is a near-native state that we can inspect now with an electron microscope. Now, this can be done with any thin object. For instance, this suspension of cells is quite thin. Also, you can take any solution of a protein, for instance, and spread it into a thin film and plunge freeze it. So here again is the picture of the 20S proteasomes, over here was the negative stain image, that I've shown you before, showing you how you can see it's barrel-shaped, but now on the right, I show a picture of similarly purified 20S proteasomes, but in this case they were spread into a thin film across an EM grid and plunge frozen and imaged in this frozen hydrated state. And so again, you can see the details of the barrel-shaped protein being a stack of four rings. And so here's a side view and then here, for instance is a top view showing the view down the barrel. And you can even see there's a hole in the center of the barrel, as we know from X-ray crystallography. And so, in this cryo-EM image, there's is the possibility of eventually visualizing the secondary structure or even atomic structure of the proteins. Because the proteins are in their native hydrated state. We still have the problem though, that we can't resolve those details from a single protein, because the number of electrons that it would take to resolve those structures would destroy a single protein. And we'll talk more about that problem in a second. So plunge freezing is a powerful strategy to preserve biological structures in a native state. And it works more easily for samples that are already thin enough that they can be placed directly into the microscope. Remember that means thinner than about 500 nanometers or less. Plunge freezing can also be used to give us views of parts of cells or other objects that are larger than 500 nanometers. For instance, even large cells can be grown on the surface of an EM grid. In this image, this is an SEM image recorded by Jeurgen Plitzko. We see a large mammalian cell growing on the surface of the EM grid. You can see the quantifoil holes of the EM grid over here and the basic structure of the large grid bars of the EM grid here. And so, over these quantifoil holes, a mammalian cell has grown and the lump in the middle is the nucleus. But we can see that this cell type flattens out, so its periphery is very thin. And in these regions, it can be directly plunge frozen and imaged. But what if we want to view something inside the thicker regions of the cell that here appear as a lump. We can't do it directly, because the electrons will all be scattered away if we try to image directly through that thick material. This is a figure from a paper from Alex Rigort and his colleagues that was published in PNAS showing this process. So here you see a cell, this is the main body of the cell growing on an EM grid. See the quantifoil holes in the EM grid here. So this is the main body of the cell growing on top of the grid. And the goal is to isolate a very thin layer of material through the middle of the cell. And so they marked using software in the Dual Beam FIB-SEM, they can mark a region of the sample that should be ablated by the ion beam. And so they mark that region and this region here. And then they use the ion beam to just ablate away all of this material and all of this material. And as a result, you get something that looks like this. Where you have quite a bit of cellular material, material over there and quite a bit of cellular material over here. And these just have a very thin, what's called a lamella. A cellular material through the middle. And if you were to rotate that 90 degrees, it would look like this. Here's the cellular material on the right. Here's the cellular material on the left. Here's the very thin lamella of material right through the middle. This is looking at it essentially form the top and then this is thin enough to record images through with a transmission electron microscope. These are two images that illustrate the advantages that FIB Milled lamella exhibit over cryosections. Both images are of, dividing yeast cells. And so, for instance, in the FIB Milled section, this is the yeast cell wall over here. And so the yeast cell goes, you know, covers this entire area and it's dividing and so here's the nuclear compartment of one of the daughter cells and here's the nuclear compartment of another one of the daughter cells and they're connected by a thin neck of nucleus here. And coursing through this thin neck of nucleus are microtubules that are part of the mitotic spindle that segregate the chromosomes. And so in this FIB Milled lamellum, you can clearly see the two membranes of the nucleus. These are nuclear poor complexes that exist in that nuclear membrane. There's others on the other side, the microtubials are very smooth and clean. In addition, you can see tubular vesicles as well as spherical vesicles and many other details about the cell. Now we compare that to a similar dividing yeast cell that was cryosectioned and then imaged. So here is the cell wall material and the edge of the yeast cell goes like this around this region. And here's that thin neck of nuclear compartment that goes between the nuclear compartment of one of the daughter cells and the nuclear compartment of another one of the daughter cells and here you can see microtubules extending through the neck, just like in the FIB Milled region. However, here you see that the membranes are rippled and they're slightly discontinuous and so that looks a little unnatural. You can see that the microtubules are not nearly as continuous and cleanly visible this is a granule and you can see that it looks somewhat compressed in this direction, it was probably spherical, but got compressed by the cutting. Here again, this is another probably spherical liposome that looks like it was compressed. And if you look carefully, you see evidence that the, the cutting blade was going in this direction. For one thing, here are the crevasses on the top surface of the section. They're not visible throughout, because it's a slice through a 3D tomogram of this cryosection. But on the edge, you do see the crevasses here that also tell us that this was the cutting direction. Then you see compression in that direction and you see artifacts in the membrane structure. So clearly, the FIB Milled lamilla better preserve the structure than cryosections. So, as you can see, there's a myriad of ways that biological material can be prepared for transmission electron microscopy. The key is always to stabilize the material, so that it can be inserted into the ultra high vacuum of the electron microscope. But in addition, if stains are used, they can enhance the contrast of the objects inside and a higher dose can be used to image them. But some resolution is lost, because then your imaging stains that simply decorate or coat or form a replica of a structure rather than imaging the structure itself. This is a flow chart that summarizes many of those techniques. Now let's start with a sample at the beginning. And at the end of the chart we see there's two basic kinds of electro microcroscopy. There's conventional room temperature electro microcroscopy where the sample is at room temperature when we stick it into the microscope. Then there's also Cryo-EM. Where the sample is going to need to stay frozen as we insert it into the electron mic, microscope. Then we further classify the techniques as whether they happen here. These are processes that occur at room temperature so this is room temperature processing steps. And these steps are all steps that happen at very cold temperatures, so we'll call those cryo-processing. So perhaps the simplest path is to start with a sample, and simply add a drop of negative stain and let it dry. This produces a room temperature sample ready for microscopy. Traditional thin sectioning EM proceeds by taking a sample and chemically fixing it. And then dehydrating it, plastic embedding, thin sectioning, staining, and then you can stick it in the electron microscope. The Cryo methods begin with the sample, and then there's some kind of Cryo-fixation that is used. There's a lot of different kinds of Cryo-fixation. We talked about high-pressure freezing, where pressure is applied to prevent ice crystallization. There's also slam and other freezing techniques which I didn't bring up. And finally, there's plunge freezing, which we did describe. Samples that have been high pressure frozen, slam frozen, or plunge frozen, again there's a variety of next steps that can be taken. Following the high pressure freezing, samples can be freeze substituted, dehydrated and fixed, low temperature plastic embedded. And once they're embedded they can be warmed to room temperature, sectioned, stained, and then inserted as a room temperature EM sample. So this is the High-Pressure Freezing Low Temperature embed route to a room temperature sample. These kinds of samples can also be freeze-fractured, freeze-etched, and other techniques we haven't described yet. And eventually these also lead to a room temperature sample, ready for insertion in the electron microscope. These samples can also be sectioned at low temperature while they're frozen. They can be cryo sectioned. And then they are thin and frozen, and as long as they're maintained in freezing temperatures, they can be inserted into a Cryo-EM and imaged in a frozen hydrated state. Finally Plunge Frozen samples, if they're thin enough, can be immediately inserted into the Cryo-EM and imaged. And finally a Plunge Frozen sample that's too thick to be imaged through can be FIB milled and then imaged in the Cryo-EM.
