hi my name is evan nogales i'm a professor of molecular cell biology at uc berkeley a howard hughes medical institute investigator and a senior faculty scientist at the lawrence berkeley national lab and today i would like to give you an introduction to what is my favorite visualization technique to see cellular of molecular details in biology this is electron microscopy in my lab we use this method to visualize processes of cellular self-assembly and molecular machines that are involved in nucleic acid transactions and i will be using some of these as examples i'm going to start by giving you an introduction to the physics behind how electrons interact with mata how the electron microscope is able to generate contrast and visualize your sample and then give you examples of biological um molecules and cells uh and how the images are then generated how they look and how we go into further processing to learn more about a structure and function in cell biology so the first thing that i would like to tell you is that electron microscopy comes in two very different flavors sem or a scanning electro microscopy is a method that uses focus beams of low energy electrons to raster along a bulky object and give you a low resolution rendition of the surface of the object so this is an example of such an image is a crab larva and it looks very pretty because it has added false color but there's no electr there's no color whatsoever in electron microscopy so that's the first thing that you have to remember this is more like the real image in an electron microscope let me give you another one this is of dust mites these are nasty things that cause allergies certainly they do on me and i want you to pay attention to the scale bar here which is 200 microns this is about the size of of these organisms you can certainly crank up the magnification in this type of microscopy and get to visualize individual cells so this is an image now of a mouse oviduct and this kind of tissue has lots of cells that have this uh celia that are used to create fluid motion and allow the sperm and the egg encounter one another notice that the scale bar now is 200 microns so we're really seeing cellular details right now i would like to concentrate from now on in the other electron microscopy technique which is transmission electron microscopy or tem this uses very different principles and what it gives you is uh projection images at much higher resolution of thin samples that are introduced into the microscope and there's possibility here to get even to atomic resolution as i will tell you in a second so this particular example um comes from a section that was taken from the flagella of any sperm the one that could have been coming up the oviduct that i showed you before so in this section what we're seeing is the axoneme a structure that goes all the way along the flagella is made up by microtubules which are organized into this beautiful uh structure now microtubules are made by self-assembly of the protein tubulin and you can actually purify tubulin typically from a male in brain like cow brain and reconstitute the process of microtubule assembly in vitro in the test tube and this is now a tem image of such individual microtubules and this image now contain information at the atomic level the scale but that in the image that i showed you before was not microns was nanometers and what you see here the thickness of one of these microtubules is 250 answers or 2 25 nanometers in fact this methodology has been used to obtain information on the structure of the tubulin protein itself and what i show you here is a density map in blue um inside the protein molecule where these yellow lines represent the polypeptide change going along it and every every one of these squares in blue correspond now to one armstrong so the resolution here is much higher is at the atomic level so i'm going to give you more examples from microtubules which are one of my favorite biological samples in a minute but before that i want to go back to very basic principles of how electrons interact with mata in the transmission electron microscope electrons have very high energies of the order of hundreds of kilo electron volts that means that they are moving at close to relativistic speed they're moving so fast that sometimes they basically go through mata without even noticing so in here what you can see are electrons going past an atom and some of them just go through and simply don't interact they don't see anything some of them and these are very important for generating the image are gonna go through and bounce of the nucleus of the atom and be bent this is a process of elastic scattering in which electrons don't change energy don't change at speed but change direction and this is what we call the good scattering and it's going to be used to generate contours in the image in the tem unfortunately there's another process of a scattering the baddest scattering and these are electrons in your primary beam generated in the microscope that go through the atom and interact with electrons in your sample and in doing so they lose energy of their own and they pass it to your sample this inelastically scattered electrons are going to only contribute to the noise in your image while at the same time really damaging your sample okay so the tm um is going to be able to visualize your sample by using these scattered elastically scattered electrons to generate contrast in the image and the way this is done is in two different manner um the first type of contrast is called amplitude contrast and is the one that is used to visualize sections of cells like this one this is a section through the root of a mace plant and you can see the variety of internal organelles in here amplitude contrast works very much like an x-ray that you get at the doctors where more x-rays are being absorbed by your bones that by the rest of your soft tissue so the bones appear darker in the image um actually what i'm showing you here is the positive of the image what the doctor shows you is the negative which looks like this okay so in the microscope in the tem electrons can be either absorbed by the sample or otherwise they can be elastically scattered and then we can remove the elastically scattered electrons but means of an aperture the aperture is placed after the objective lens and what it does is it allows the unscattered electrons to go through but it blocks the elastically scattered ones and that generates contrast in the image basically making regions where lots of scattering happen because there's more density appear dark and regions where there was less scattering appear brighter this type of contrast is great to visualize sections of cells but it falls really short when you're trying to get high resolution information on proteins and other macromolecular complexes so for that we need phase contrast and this is the contrast that is generated in these type of images now of purified protein and protein complexes in this case the objective aperture of the microscope is utilized to make the scattered elastically scattered electrons and the anascato electrons interfere generating contrast in that manner this is based on the principle that relativistic electrons basically can be seen as waves and that when they go through mata the wavefront will uh suffer a phase shift and this is what will ultimately cause the interfere when interference would mix with the unscatted wavefront i'm not going to go into any more physics rather i will now show you how an electron microscope looks like okay this is what i would call a middle of the range electron microscope it start the whole story starts up here with the electron gun which is where the electrons are produced they shoot down the column which is maintained at very high vacuums so as to minimize the scattering of electrons by residual air at each stage you have electromagnetic lenses that are used to deflect the path of the electrons earlier on there's two condenser lenses and these control the illumination on the sample how bright and how large is the area that is being illuminated and then comes the most important lens in the scope which is right in the middle is the objective lens this is the one that will combine the scattered and non-scattered electrons to give you contrast in the image and the other thing that happens right here is that's where the sample goes in in this case this is a cryo sample that is being maintained at liquid nitrogen temperature by being in contact with this dewer with liquid nitrogen so after the objective lens come a number of intermediate lenses that are utilized to change the magnification from 50 times to something as high as 400 000 times the image can then be observed in a phospho screen or directly on a tv and ultimately record it for further data processing either on photographic film or in a ccd camera the one thing that i would like to show you is an electron microscope grid is right there on my finger this metal grid is covered by a thin layer of carbon and then the sample goes in there and we lead we need extremely little amount of sample so we use say for purified complex uh protein complexes concentrations that are on the nanomolar sub sub micromolar range and we used a very small drop the size of a small tear that goes right in there and then gets plotted to a thin layer before it's very quickly frozen the grid um all of this is done on the liquid nitrogen or nitrogen gas and then uh in a holder and then is introduced in the electron microscope but with very very little sample we're going to get still get millions of occurrence of the complex that we are interested in and take many many images and hopefully get to the structure we need i can often ask what kind of resolution can the tem reach and in fact microscope like the one that i showed you is capable of obtaining images with atomic detail this is such an example these correspond to a thin nanocrystal of silicon where each one of these atoms corres each one of these dots is are actually atoms that have been visualized in this image in fact a state-of-the-art electron microscope can reach resolution beyond a single on single armstrong so very high resolutions are achievable as long as the sample is not radiation sensitive so this sample was able to withstand thousands of electrons going through it in each square armstrong in the sample without damage unfortunately that is not the case for biological material which is extremely sensitive to radiation it was only a few years ago that people thought that high-resolution information from biological materials would never be rich because the sample will vaporize before images were finally collected if that had been the case i would be very sad would not have a job and i would be not talking to you today but fortunately there are more than one way in which we can trick nature and obtain high resolution information from our biological samples so let me review with you what the problems are that are unique to biological organic sampling first of all all biological material lives in aqueous solution and by definition they hate the high vacuum in the column of the electron microscope the other thing is that the atoms that biological materials are made of nitrogen carbon oxygen have basically the same scattering power as the water that is surrounding them so they basically have very low intrinsic contrast and ultimately and most importantly they are very radiation sensitive with when inelastic scattering um intra occurs the sample gets ionized generating radicals that then move around the sample and break all the bonds and basically make the whole thing explode okay so how do we overcome these problems there's two solutions the first one to occur historically was a negative staining in this case your sample is embedded in a low concentration of a salt solution of an a very heavy atom typically uranium the sample is embedded in this in this solution the solution is then dried to a thin layer and introduced into the electron microscope because now there's no water there's no problem with the vacuum the heavy atom that uranium generates very high contrast so we could over the second problem and because now what you're imaging is this cast generated by the stain rather than the protein you also reduce the problem or radiation damage the protein may vaporize but as long as the caster still reproduces its shape we are okay so the big pluses of this methodology are the high contrast in the image and the fact that is fast and easy so berkeley undergraduates can come to my lab and in a few weeks they're ready and taking beautiful images using this methodology now there are minuses of course the miners the big minus is that artifacts are really possible this is due to the fact that in some cases the stain cannot penetrate inside the protein or cases where as you dry the stain the protein the protein structure may collapse even if you have very good preservation in some cases the resolution is always limited it's limited because as you dry the stain it forms little grains and thus the ultimate size of anything that you're going to be able to see which is typically about 15 angstrom so the solution to it to this the second solution optimized solution is to look at unstained frozen hydrated samples this is what we call cryo-electron microscopy in this case the sample is embedded in its accurate solution where it is happy but then is very quickly frozen is frozen so fast that the water molecules don't have time to reorganize into a crystal into ice and therefore remain amorphous we call that vitrified water this um to achieve vitrification the samples have to be frozen very fast typically a million degrees per second and then kept at very low temperatures the temperature of liquid nitrogen if you do that this sample can go into the electron microscope without evaporation of the water so we avoid the problem of vacuum all together so the sample is hydrated but in a solid state that can withstand the high vacuum now because there's no stain these samples do suffer from low contrast and we're going to have to overcome that by all the means i'll tell you about that later radiation sensitivity is limited because now the very low temperatures mean that radicals that were generated through the process of inelastic scattering are not able to move very fast so that radiation damage is minimized not eliminated but reduced so the pluses of this technique is that the preservation is extremely good because basically you have preserved even the aqueous layer that surrounds your protein because there's no stain and there's no graininess high resolution is in principle achievable in fact in some cases if you have the right experimental setup you can even obtain time resolution you can time a certain biological process to be triggered during the process of vitrification and trapped intermediates now minuses this is a much more technically demanding technique so undergraduates in my lab rarely get to a point where they are feeling very comfortable about doing uh crying it takes many months if not years to really master the other problem is the contrast as i tell you there are ways of enhancing the contrast in the electron microscope but they always tend to come as a price so mostly we deal with that computationally the other problem is that although we have minimized radiation and still the sample remains sensitive so we have to use um low doses typically 10 to 20 electrons per amps from square and that means that the no the images are going to be very noisy okay so let me give you an example how the the same sample the same biological material looks like in negative stain versus crayon so what you see here on the top is an image of a microtubule that is surrounded by rings that are made of a kinetochore protein the kinetochores are the structures by which microtubules interact with chromosomes in a process called mitosis by which genetic material is separated so here is that sample in negative stain this is urinal acetate that is generated to use to generate these very high contours notice that the proteins appear white while the stain around it appears black the image behind is exactly the same sample but now what you're looking at is just the contrast of the protein on a background of water and the the image appears a lot cleaner because in here we can see every individual protein even those here in the background that have not self-assembled into these beautiful structures while this in here is basically invisible on the other hand what we have really present very beautifully in the cryon image is the cylindrical shape of the microtubule and the circular shape of the ring which allow us to obtain ultimately the structure in in high detail and with high reliability okay so let me now go back to the electron microscope to show you what is a true a state-of-the-art tem machine all right now this beast is what i would call a state-of-the-art transmission electron microscope you can see that the column is both longer and wider this is because the electrons that are being emitted by the electron gun have higher energy as they are moving faster they need bigger electromagnetic lenses to deflect them this microscope has two special very unique things one is the sample goes here this is what we call the stage and this sample is being maintained at liquid helium temperature that's very much close to absolute zero or minus 270 degrees centigrade okay so that reduces radiation damage and also the whole mechanical stage makes this sample very very stable and it makes a difference in the sample really doesn't move when you are taking the picture now the other thing that is very important in this microscope and the reason why it is so tall that i have to stand on a ladder is that it has an extra piece right here this is an in column energy filter it works very much like a prism but for electrons it spreads them out in a rainbow depending on their energy and that allows us to filter out the inelastically scattered electrons that are contributing only to the noise in the image this is particularly important when the sample that you're looking at a thick uh sections of thick cells where the signal is going to be very low and the amount of inelastically scattered electrons is very large so these were allowed to clean up the image and be able to visualize things that otherwise will be invisible okay this is a good time now to recap and think of the basic principles of how to generate images of biological materials in most cases we start with a purified sample of your bio of your biological material of interest this one is deposited on a substrate in the en grid that i showed you before typically covered with carbon and is either embedded in negative stain or in a thin layer of vitrified water then we pass electrons through it some electrons go right through and others are elastically scattered and will be the uh interaction of the unscathed and scattered electrons that will give you an image in the electron microscope but remember although we start with a three-dimensional object what the image in the tm gives you is a two-dimensional projection of the object remember this is not a surface like an sem it is a projection of the whole structure but now compacted if you want into two dimensions things are really worse than that because the radiation sensitivity of the sample means that we're putting very few electrons to generate the image and the image is really noisy so this is the true data that we have to deal with from here from these noisy two-dimensional images we need to get back the three-dimensional object in great detail so how do we do it that is the details are going to depend on the type of sample but typically involve a process by which many images of the object in the same orientation are identified aligned and averaged to recover the signal so that now we have things that looks look more like that if we can get these type of images now but of the object in different orientations then they can be combined combined for as long as we find out the relative orientation between them to move from 2d to 3d and recovery structure okay uh this process is what we call reconstruction and how each one of these two steps are carried out depend very much on what type of sample do you have okay so one type of sample that is ideal but comes very rarely is that of two-dimensional crystals of proteins in this case the protein is arranged in a single plane in a in an ordered lattice that can stand for several microns with a thickness that is just a single protein okay these kind of samples always falls in the same orientation in the grid so in order to get three-dimensional information is absolutely required that you do what we call tilting this means tilting the sample with respect to the electron beam so that we can generate different um views of the object okay this tilting process is actually experimentally very complex and difficult but when the data is collected the computational processing is very simple and it actually allows you to get to very high resolution fairly fast this is because the image of this ordered array contains very high resolution information as can be seen in this electron diffraction pattern from such two-dimensional protein crystal which is 10 to about 3 ounces of resolution another type of sample that is really very helpful and great for doing em are helical arrangements this can be naturally occurring or they can be artificially produced because in in a helix the molecule is in different orientations as as you process you know as you move through the helix you get different views that are related by the geometry of the helix so no tilt it is needed and you can actually obtain a full three-dimensional reconstruction from a single image although initially it may have low resolution now uh this type of methodology is able to get between uh medium to high resolution meaning between 10 and 3 amps of resolution and like for crystals the order in in these structures means that in fourier space if you want in the diffraction pattern we have reflections that are well separated and we're filtering i'm not going to go into details but the filter is equivalent of an averaging process so the 2d uh classification and alignment and the three reconstruction are very trivial computationally for both of these two samples however the most general type of biological sample is not going to be a two-dimensional crystal and it's not going to be organized into us into a helix in that case the type of reconstruction that we do is called single particles typically these objects are going to be randomly oriented in your em grid and no tilt will generally be needed the type of resolution that you get is going to depend on the type of sample for objects that don't have any internal symmetry and that may have floppy regions the resolution may be very low the order of a few uh nanometers while um for objects with internal symmetry is the case for viruses the resolution can be very high all the way to three or four armstrong in these cases uh but there's no supramolecular arrangement the computation is really heavy it takes a big toll on the data processing so let me show you some examples let's go back to microtubules microtubules are an example of cytoskeletal self-assembly into helical structures as i told you microtubules are made of alpha beta tubulin which are this represented here by these dark and light um cubes they associate a longitudinally making what we call protofilaments and these associate in parallel making the the wall of the microtubule from images like this of this structure using helical reconstruction procedures it is possible to obtain structures like this where each one of these um correspond to a tubulin molecule and you can see details on the secondary structure the architecture of the molecule one at a time it so happened that the case of tubulin you can trick it to self-assemble into something different where protofilaments is still formed but they are social in an anti-parallel fashion where the structure doesn't close into a tube but rather it grows into what can be considered two-dimensional crystals these are the ones that produce this beautiful diffraction pattern that i show you due to the high order um in the in this polymer and from here it is possible to obtain atomic resolution information and that's where um ribbon diagrams like this that now describe the path of the tubulin chain could be obtained i just want you to notice that this was obtained in the process of taxol which is here shown in yellow this is an anti-cancer drug that is used to bind to tubulin and stabilize microtubules but microtubules very stable and that stops the process of cell division and it stops in particular cells that are dividing very fast those being cancer cell typically microtubules are very highly dynamic and microtubules have been the object of crying study to describe actually how the process of assembly and disassembly take place so what i'm going to show you now is a short animation that describes a very graphic way how we think microtubules and the whole process of assembly and disassembly based on cryo-em structure of the intermediaries that are generated in the process so this is a microtubule that has reached a critical estate um where it's going to lose its stability and it's going to start depolymerizing this is the tubulin structure that i showed you before so that you have an idea of how it arranges into the microtubule the microtubule breaks down actually by peeling and curving of individual protofilaments the pills are normally very short-lived they break apart and they depolymerize into individual subunits but we were able to trap them by biochemically and in order to obtain the structure of tubulin in that conformation and what we found was that tubulin subunits are not only interacting with their kink but they are kink internally and that um is what makes it impossible for them to remain stably in the microtubule as a molecule of gdp is exchanged from a molecule of gtp that re-energizes the tubulin molecule and straightens it up and allows it to now form both longitudinal and lateral contacts in worries and we believe and a structural intermediate in the process of assembly that is open and outwardly curved we could again stabilize that polymer but means of low temperatures and a non-hydrolyzable gtp analog and this is the structure what we saw was that protofilaments here are paired up and within one pair the interaction are just like protofilaments in the microtubules but between pairs this interaction has rotated and as this thing grows it eventually starts rotating around that special interface so that it closes into a tube in a process that can be very highly cooperative by zipping up of the tube as the protofilaments is straightened so typically you would have a microtubule that is growing by addition of tubulin subunit into an open sheet that then closes into a tube and eventually this microtubule will grow will reach a critical step and they will start depolymerizing and assembly and disassembly will constantly occur as in the cell okay so i showed you examples um using tubulin uh of how um helical reconstruction or two-dimensional um crystals are used uh to obtain high resolution information on sample but in many cases we have to rely on single particle techniques because these higher order structures are not available so let me very quickly go through uh the processing that will have to take place in a single particle project in order to get to the final structure to start remember that uh we have our sample em embedded again a purified sample are molecules embedded in either a stain or vitreous water that the em image gives you a two-dimensional projected projection that is actually very noisy because of the low dose that we can use now from here what we will do is we'll visualize each one of these occurrence of our say protein complex and we'll pick them out and generate galleries like this that shows our different molecules these are showing the molecule in different in-plane orientation but also different views so what we go what we do computationally is we go through a process of aligning these images to each other and then classifying them so eventually we put everything that has shows the same view in different classes and now these are ready for averaging and the averaging will give us now enhanced views of each of these orientations of the molecule okay these now have to be related to one another and this is a very tricky step that i'm completely going to forget about for now but ultimate is can be very computationally uh involved but ultimately if the relative orientations of these of these different views are obtained um we can go and reconstruct the object in three dimensions okay so let me uh now illustrate how do we go from the two-dimensional images uh that we know are related to one another by defined angles to obtaining the three-dimensional reconstruction we do that by something that is called back projection so imagine now this is a very simple example where your object your molecule is made up by these three circles okay so when you pass electrons through it you generate a two-dimensional projection that looks say like this okay and of course um you're gonna have this object in different orientations in your em grid which means that when you take different images what you get is um different projections that look distinct and that by some means you're able to um place one in relation to the other by finding the relative angles this is tricky to do but once you've done it the way to obtain the reconstruction is to back project what does that mean you take each one of these projections and you smear it um and you see how all of them intersect reproducing the object so this i have another movie that is a little bit more fanciful because uh patrick's and patrick's day is is coming the day that we're filming so this um this is our object and what i want to illustrate here is how as we use more projections that are equally distributed uh we get more and more accurate representation of the object um so this is a movie in which now projections are being added and the intersection is giving rise to um to the sleeve now in more and more detail okay um so let let me now illustrate all of this with a real project this is our study of the exosome which is a molecular machine that is involved in processing rna and in some cases in degrading rna and the exosome in this case from yeast from budding yeast was purified and each one of these blobs that you see here correspond to one exam one complex one image of the complex and the complex is randomly oriented in here and this is actually by the way a negative stain um image so all that i showed you up to now was cryo-em but this is an example of a negative stain study so uh if you go and pick up individual particles this is how they look like this is a gallery they're pretty noisy but going through the process of alignment and classification and averaging you get now images like these that look look a lot more well-defined so the tricky part which i'm skipping is how each one of these images are related to one another but once that was sorted we were able to obtain a three-dimensional reconstruction just to tell you we obtained two reconstruction one of the full complex that is shown here and one of our core element in the complex which structure had been obtained at atomic resolution by x-ray crystallography of the human homolog when we subtract one from the other we get the core in blue and this extra region in yellow which happen to be the one that has um the biochemical activity um the site that actually chops the rna now what is shown here is now the crystalline structure of the human homolog of the core domain and what you see here uh in yellow are pieces that were taken from homologs found via bioinformatics and what this allowed us to do was to create a pseudo-atomic model of how the top and the bottom part of this structure interact now this is actually a very common type of methodology we refer to this as hybrid methods and it it involves the docking of crystal structures of components into the low resolution structure of the full functional complex and this is something that not only tells you how good your structure is but also give you new information on say interfaces how this bottom part of the um the top and bottom part interact which elements uh are involved in in that interaction and in this particular case it gave us the path of the rna of the rna by aligning the uh cavity in the top part with cavities that exist in the active region that lead you all the way to the active site okay so no matter whether the molecule that we were studying was in a two-dimensional crystal in a helix or was a single particle where many copies were utilized and combined to generate the structure we were always looking at things where there were many copies of identical copies of an object but what happens when we're interested in something where not two are the same like what we're interested in organelles or cells what do we do here in that case what we utilize is the method of electron tomography in electron tomography the basic principle is all the views that are required to obtain a reconstruction have to be taken from a single object not for identical copies but from a single object so in here again the idea is we have a very unique sample for which there's no identical one say an organelle of a piece of a cell and what we're going to do is we're going to take many views of the object by taking this object and tilting it and always looking and shooting grabbing images from the same object okay and these the images will be obtained by tilting very gradually typically about one degree although how fine that division is made depends on the size of the object uh in here how these images are related to one another is very easy it's just determined by you you were always looking at the same object and you were the one uh telling the microscope to tilt by a certain degree so computationally this is experimentally and computationally is very easy to obtain a reconstruction which is in this case is again done by back projection the difficulty here as you will see has to do with interpreting these images which tend to be noisier and objects that are really really very complex so we have utilized this kind of methodology in my lab to study yet another another self-assembly system and that is the one on septins uh which are proteins that self-assemble and make filaments that actually lines a particular site near the membrane in the cell are the position where cell division is going to take place we study septums in the organism where it was first discovered with it which is the body in yeast and what we are interested in when we look at these cells is just the particular region here where our filaments are formed and where we want to see how they are organized and how they're interacting with the cell membrane okay so the first thing that we do is to collect a tomographic teal series where we place the object in the electron microscope decide what is that we're gonna shoot at and then take images um once for every tilt of one degree and here these are these images are showing just one right after the other so this doesn't correspond to a reconstruction yet this just shows you one of the other the images of a very thick section where it's very very hard to determine what is what is in there so after this still series are used in back projection we can generate the reconstruction and i'm going to show it to you as a series as a content series of a slice is going in and out several times uh in the reconstructed section okay so this is the section we started at the edge of the section that we're going through and i hope that you can see now that we see in much more detail as we go as as we're going each one of these speckles correspond to uh ribosomes and there's many of them in the cell what you see here is the double membrane of of a nucleus um there's a lot more in the membrane here and of course uh right by the edges is where we're going to see uh objects of interest which are this self-assembly of septins into filaments around the membrane so you can see the complexity of reconstructions like this there's so much going on so in order to be able to look at it at once what we do is we simplify this image but just utilizing simple surfaces and lines to uh trace through the surfaces of the plasma membrane of the nuclear membrane or the filaments are that we we can trace from one section to the other and we get uh renditions like this by what we call segmentation so this is now a very simplified view we eliminate the things that we were not interested on like all of the ribosomes and what you see here in yellow is the pump plasma membrane is very curved because that's the side of the of the butt neck where the septation the division of the mother and daughter cell is going to take place this section included the nucleus that is also in the process of dividing with microtubules shown here in red that are pulling chromosomes apart there is more membrane that is internal that is shown here in canon orange uh actually the thing that we were interested in looking are these filaments uh that run in a number of directions they run around a circle if you want in the butt neck but they also run between our daughter and sister cells so they're the ones that we're showing here in green the ones that we're showing in blue and interestingly there are also small filaments that we see in that are shown in red that are connecting the membrane to this filament system so just as a final note imagine um all the information that is concerning containing the tomogram that i just showed you a minute ago where we only concentrated in this small section uh it would be great if tomograms are made available publicly available just like crystal structures or electron maps of reconstruction so that anybody irrespective of what you work on can take can make use of the image to follow and track the object of their principal interest okay so this is the introduction that i wanted to give you of this technique and i hope that in this brief time i gave you an idea of the general generality of how applicable uh this method can be all the way from individual molecules to visualization of the cell and what i haven't time have any time to tell you is that um this method is far from being totally optimized and that there is a lot of development and improvement in the pipeline that is going to allow us to get not only higher resolution but study even more systems that right now remain really challenging so by the time someone like you is ready to use this technique things would have really moved beyond what i showed you today so i really cannot wait for that moment myself