DevoWorm Research Presentations, 18 Oct 2016

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From YouTube: Yorgos Nikas talk

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Yorgos Nikas (Athens Innovative Microscopy), talk to DevoWorm weekly meeting, October 18, 2016. Title: "The Microscopy of Mammalian Embryos and Reproductive Biology".

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Okay, look again Hey.

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Nice to see you so I will save the screen. First, please let me know if that works well with you, Start screen say: is it now?

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Can you see now my screen yet.

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So the toka I will I will show you with a moment because I have a problem now. What is he? Okay from.

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Gametes to the blastocyst changes in cell surface morphology- you know, I am a morphology. I am a doctor, basically and I understand only morphology molecules, I, don't understand. So it's the only chance for me to do some results to see the same of the things and I'm fascinated with microscopy scanning electron microscopy and here I will start the story on the human life from the beginning. In a way that here, you see an ovum human egg, which is at the time of ovulation, is surrounded by the human cells called coronary da da this cells.

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Actually, they feed here is a graphic. This cells, they communicate through projections and meet the projections from the oven and communicate through gap junctions, and it is the way that the egg is loaded by all these substances nutrients and the cell machinery components in order to grow and to be able to sustain the new life and I think, actually that the perfect round shape of the egg is due to this action of at least 5,000 tiny cells, normal cells. But the oven is big that they act all over and they act equally.

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So it gets this round shape, it's just a thought now, but what happens a little bit before ovulation, because the human Collier holotropic human barometer often is secreted the la Sol, and then this.

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This this protection I saw you before they withdraw both sides and they live empty spaces. Here by the way, is the egg cell? It is the zona pellucida by blue and inside is the ovum itself, and here is the first polar body and no one knows how the sperm really makes its way through the humans, karana layer and the seat and elastic zona pellucida in order to fail to enter and fertilize the egg, because to recognize its melt lemma, we call the membrane of the but I.

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Haven't people think that it makes its way between this coronary the other cells and then dig a channel inside the zona pellucida to meet the problem, but I will show you evidence that the sperm action occurs through the correlations not between through through the cells and it uses. One of this force that is left by the the empty space is left by that neutral withdrawal of the protections to to mix the so next slide. Now. Yes, this is now karana cells.

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This is the zona pellucida in yellow, and these are the protections they will deep inside and now they withdraw.

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This is an oven that way partially remove the karana cells to expose the zona pellucida. Here you see.

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I'm sorry I will start a game for because I talked about the right menu here like this.

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By scrolling, it doesn't move to use this next anyway. This is a detail of the previous image of of here sample and we see in detail how the zona pellucida is made from three four layers of rings.

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Look like rings here and in this light, I put together in the same magnification amateur and on the bottom and the immature egg, and you see in the immature egg you that this pores are very different because it didn't have the time to expand, and this is the mature egg with expanded pores, and this ring shaped structures is very important for fertilization. I. Think now you see another SEM image. It is full of little pores here.

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Let's talk about the spermatic bit which, because it is with them to ignore the the malefactor no and this is spent traveling its weight to throw the fallopian tube to meet the egg. This is a perfect wave. If you follow this, there was another call here, but the agency cut it because it it found it confusing, but actually it makes seven curves of an harmonic wave, it's very difficult to get the sperm so active to to kill it so active.

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Usually it gets like a spaghetti like this one, not because it has the time to do some movements and before dying.

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This is now the fallopian tube here and I have put an egg here too, to solve the actual science of the iPod I wouldn't find in this, and this is the fallopian tube, and there is here sperm. If you see inside here, there is sperm, it's very rich, this visual, it's the light at the bottom of the signal, like the signal. This is perma again in the fallopian tube.

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Now this is the at the ovulation it has, as I mentioned before it has the corona cells around and the zona pellucida in yellow you see inside and the sperm case, to pass first discriminations.

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The way this passes is a mystery, but if you see this in this life, this Fermi is a has lost the acrosome. The acrosome is the cup here, which should be lost in contact with the zona pellucida. This prematurely lost the aperture, it underwent the acrosome reaction and the hyper activation, which is needed to pass through the zona. But here it's pretty much Larry. It happened you can tell it is reflective, aided by the shape of the tail, and actually it is stuck here.

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One coroner says it would not go further. Other sense, other Spencer goes further.

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It looks like it penetrates the coroner cells and such figures that has been interpreted like Thomas I toss this off spent by coroner cells, which I don't think it's to this because the sperm does is not damaged. It looks very active the city's figures. It looks like it enters here, the corona cell one another here and if you do a transmission electron, you see here that it looks like it seems freely. Within the cytoplasm of a corona cell. There is a hollow around.

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There is no evidence of phagocytosis, and actually the sperm is used to be in in the cytoplasm of other cells because it grows in the sertoli tubes in the industry. In very sorry, in the recall, the demise of.

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Inside the the spirit of Genesis is happened within the secret of sertoli cells, so the sperm is used to be inside somewhere else. You know do it and when it is free, it gets anxious to get sample in you know.

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But let's talk about the next layer, which is the the zona pellucida, is very important for the selection of the sperm also, and the also it blocks polyspermy in the way. I will show you very soon and the zona pellucida has to set off before implantation.

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Now, no one knows how the spam really penetrates. This elastic called the zona pellucida and they have developed two hypothesis, the mechanical that is primarily mechanical forces and the enzymatic that the primary role is enzymatic reactions. This I I got from Canada Massey, who was I, think the best so far Japanese, but living in Hawaii, I think to study fertilization and now I a very important physical question that, as we know, the sperm strives to enter the egg so what it doesn't turn away because I know from physics that any moving object.

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If the encounters resistance, it will turn away somehow it cannot stay but exactly opposite to circle.

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Will turn it will take an angle and then escape? So what is this that holds the spoon fed firmly there and it can only move inwards and I. Think the answer is not enzymatic or it is. The answer is mechanical, because if you see here all these drinks in the egg here, so if they must not so good- and we see the sperm that actually enters the zona pellucida through this rings.

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This suppose, if you see this image is here so must be something like like here, I put together that once the sperm enters the outer circle, it is interrupt there and can only move in whirl around. So it is a like a mechanical factor that guides the sperm inside.

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I I like to compare it with the sex. You know sex in the microcosmos, because if you see the, if you see how, similarly is the sperm with the pennies, it.

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The similarity you know, and even the head is like in the head of the penis of the and the also the acrosome reality the acrosome is is like the poster say here the to the cover and also the links of the egg as the.

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Vagina has rings.

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Here so it's like sex in the microscope, the Nitro repeats itself and at that time of capitalisation, the egg is in the fallopian tube.

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So that's what we believe at least, because no one really knows after the one sperm enters in the egg immediately. There is a like.

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Big reaction of the egg waves of calcium are are all the cytoplasm of the egg and there is the release of the so called cortical granules small cysts, with enzymes below the surface of the egg, and this has a proteolytic enzymes that they change immediately. The zona the zona pellucida from fenestrated, as you see here on the left to a compact, the ones as you see, on the on the right and actually, when I publish this. The referee told me that.

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If you notice the outer rings, if a force from outside has made the zona compact, it would affect first the outside rings. So it comes from inside.

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So the I believe that the elastic this elastic hoops as what we call the Rings may act as mechanical receptors for spent.

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This important function of the zona pellucida now is completely bypassed by the spare microinjection. You know that happens in IVF and it happens now in ninety five. Ninety seven percent of the cases they do immediately high-technology, even if the the husband has a good spell. You know this is not good anyway, and this is, we did the work with some, what one groups from France and they they had a population with some cycling, which is a cell cycle. James, doesn't.

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And the meiosis doesn't work and for spermatids remain John joins and therefore completely infantile, disguise so to carry on now to the embryo. I used the mouse model to describe what happens after the second and thirdly, which that renders eight cells in the mouse.

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Then a very important function happens, which is called confections so in compaction. Very segregation of the cells to the inner and the outer the embryo does not be starts. The first time to behave like an organism and not like separate cells and after compaction.

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The mitosis of the cells continue and the cell numbers increases, and they there is fluid through application. Fluid transport fluid accumulated inside the medulla that pushes the inner sense to one side and they will form the inner cell mass, which will form the embryo itself and outside. It remains the outer sense that the trophoblast, which will generate the extra embryonic tissues.

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We don't know we did a study to see what is the timing of compaction in the human embryo, because there were no much data about it. It seems to vary between the various species. As you see here, the bovine is later in the in the PV is even later.

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Who started from unfertilized? This is not an unfertilized egg, but I removed the zona pellucida and you can see the inside the old Emma. It's called all or Lima or vitelline, maybe the more familiar with the spittle and membra, and this is the first polar body here and we see there is a lot of microvilli here. You see plenty of microvilli and some long ones. At the area of the first polar body below the polar body lies the spindle.

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Which is because the egg is arrested, an active state so of- and this is the fusion of all the sperm with the earth and 21.8 futures woodcuts this point. But at the moment we had low resolution machines and we could not say I could not take a better picture, but they immediately after fertilization.

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The number of this methacrylate decreases dramatically and there is a theory that this microvilli works, like a reservoir of membrane components that later are used to form the pro-nuclear heads and other structures inside the sense that they need for tools to sustain growth.

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Now, with we are at the site of the two cell stage, this you never say unless the embryo gets. It is very first stage the second cell, the traditional stage. Then it is the fortune stage, nice embryo, with equal decimals. You see the zona pellucida here around quite sick, often the zona pellucida. This is electron microscope now and you can see the blossom and we call blossom and Suzanne says no. She, okay, it's the same in accident, I. Think.

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This is fortune states still, there is no science of compaction. This is a poor quality in embryo with values things on the top like spreading one cell to the other. We don't know what is the significance of this functions.

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This is eight states even more at the day. Three. This is not a. This is ten twelve cents cents day, three still no compaction and the platformers are hot together by long microvilli.

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As I told you at this stage, behavior not like a single organs, but it's just an aggregation of shells did not communicate much tree to each other, and that gives the opportunity to remove one cluster man.

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Make a genetic diagnosis to the to the embryo you see here is the opening in the zona pellucida. It is at the time we did it with acid tyrant.

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Now we do with laser, and this is an embryo which I put on the top of the needles for the tip of needles, to actually show the the size of the embryo to people that they are not with microns and all this, and they can get an idea of how little is the embryo, and this is important for people working on the ethical aspects, at least to know that the embryo is not anything with the eyes or legs or it's just an aggregation of options, and actually the term a that was valid.

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For many years was a fertilized ovum, not embryo the term embryo and the American pre embryo is more porous. The term embryo emerged after in-vitro fertilization because it was too much to say and fertilized fertilized and they say embryo and is more more important.

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Know yeah, but that created a lot of hassle in the ethical point.

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Here is the same embryo and look. There is a sperm here engulfed by one experiment, because in vitro you put medicine and maybe one passes, but it is it in the in incorporate, but this can create a big massive in genetic diagnosis. If you remove this blasphemer for genetic analysis- and there is a spanner my own on it, you will get funny results now. Look this embryo is one stage further and concerns to compact, so the blasphemer flattened to each other, but they flattened also on the apical side.

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No, they flattened to each other, but also there, and it is the first sign of compaction that this is more compacted lambreaux marula states they fall and if you, secondly, if you segregate the blasphemous, you see that there is no differentiation of the cell surface at the points of contact. There is no more long micro villi, the magrav light disappear and areas of conduct with some bubbles here, and this is more compacted marula and we kept the time- and this is a bad embryo, with a lot of what we call. These bubbles like.

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This is about the embryo, bad quality for a high and the F, and it is interesting that it fits one blossom and to the other, like this, like a toy you see, and if you segregate, you see it's like a boy's toy, it was at the feet together to one to the other. We don't know what's. The significance of this phenomenon is difficult to investigate so from the study will conclude that the human embryo and the strong contraction at the 10th of 16 cell states on the day for post insemination, in vitro in.

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Anything which is a pity now that we need a lot more information about what happens in vivo because we interfere and we do in vitro fertilization now is bad research of human embryos before it was, it was allowed and now it's bad stage.

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We did the cover of this. For because you know the the photos are are building and I. Don't know if my work was so so significant, but they did the coloring. So now we go further and now the egg is not anymore.

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In the fallopian tube.

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It is in the uterus, this is the uterine epithelium. This is an egg. This is an embryo actually because it's still enclosed on the zona pellucida, but inside must be Amarula, as I saw you before, and now. Let's talk about the oscillation I already mentioned how it happens with the inner cells, the outer shells and the transport of fluid to pursue. The inertial must one side, and this zona pellucida fails to be said before implantation and how this hatching happens.

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Look. This is a human embryo, it is they they filed and it started to hatch. You can see this part of the trophoblast protruding from the zona pellucida.

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This is the same embryo on the tip of the pin for my course. No foreign electron microscopy studies- and here is the point of cutting it's very interesting- that it is very, very little if you see how small is this opening it's very small, it's like an atomic bomb. How do you call this? You must remove of the atomic bomb, it's very dramatic moment in the life of embryo, and it needs to be a strong embryo to undergo hatching and then the first from the hatching point, which was first a little circle.

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Now the embryo managed to make a long sleep from from there and the whole pod ready. A big part of the embryo is getting out. This is now with laser. We do this with laser for blastocyst bags.

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We never let the poor embryos intentional, and this is the the holding the better as we go through suction. It helps the embryo.

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Here is the laser reaction of the laser that, if you see the same embryo here, it's the same embryo in electron microscopy and some cells are ready fried here through the action of laser. You can tell from the membranes how they are, but there is some protein around that creates this mesh, and this is now assisted fashion. We call the open hole in the embryo in the zona pellucida in order to facilitate hachi I, don't know if that really helps implantation.

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You know, because maybe if the embryo is vital, it will hurt by itself, but anyway, people do it yeah.

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This is now more advanced hunting figure of eight, because here the embryo here with color from my wife, my wife is designing, and since this one this is the employee- and this is the zona pellucida, so it could not stay longer here it wants to expand, and this is now now look now I show you open a crack open the embryo to expose the inner cell mass, and this is an embryo that looks a from outside the trap of last lecture of good, because there is here a lot of microvilli.

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There is some specifications here which I will show you later that they are important for signaling towards the uterus. But if you crack this exactly this embryo you see inside the inertial mass is degenerate. You only saved fragments and bubbles, blabs options.

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So it can be that the inner service doesn't go well, but the the trophoblast is still alive, and that gives pregnancies, but the so-called and embryonic pregnancies. Well, you get a positive pregnancy test and the sack but no embryo. It is very embarrassing situation and sometimes needs torito's.

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Now this is a good embryo, j5 enclosed in the zona we open it to expose the inner cell mass I will show you here more details. This is the inner cell mass and this year, I will show you better in color, which is there. The inner cell mass is here in red, and you see there are some dead cell debris here. Some cellular death, but not so much not like the previous one.

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There's always some some dead cells, and here is an interesting point at the junction between the trophoblast and the embryo blast and the innocent must. There is one cell that has irradiated many projections. Touching many other cells and the other cells are also radiate projection. So it seems that this must communicates very strongly, and maybe this the organizer as well.

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The organizers organizes the inertial mass. That, already from day five has some shape, maybe has up and down left and right and.

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Now another embryo was what this.

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Inner cell mass good inner cell mass here you see in light microscopy, it started to hurt so on day. Five also I open I put it first here and then arson mass goes down because of field physical law see it is a more mass at this point, so the needle attracts more the site of the inertial mass than the other, and it jumps on the middle because, okay, it just by itself, I, don't put myself there.

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I could never put one thing that is tenth of millimeter to catch it and put it there that by itself- and if you open it, there is a lot of protein in inside which you need to brush it out. And then you see then the inner cell mass here- and this is the point of cutting from the inside.

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This is a so-called blaster cell. It isn't the cavity this embryo head to to in inertial. One is here- and there is a second mass here- you see a little bit black, but maybe not so vital and after I opened the word to two inner cells, one this and the other, this one.

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Although the 2020 no and basically has a blue bottle- and you think by this picture- that it's a good to a blow with many cents- it is a bit black though, and if you do scanning electron microscopy, you see that this embryo is no good, because the cells are in bad shape. You see the plasma membranes, they are fragmented and now, finally, I would so with the best I ever, which is a big blastocyst day, six with expanded blastocyst, and now it collapse there and admit it's not perfectly.

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This is the light microscopy inertial mass here and now on the way from microscopy on the tip of the ping.

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There are many here, many.

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There are many like raffles like something just it's difficult to tell what there they are doing, and there is these were started years ago, but by in the mouse.

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This is an old publication, I found and they comment on this reference, but they say they've received like cancer cells and so on and I could not understand what these ruffles are are doing until I saw this look it. It was the the only one here. If you see here. This is what I will show you in more detail this one here. First I thought it was a rubbish.

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I didn't pay much attention, but if you see in this image here this and this- and this may be the same structures at different stages- it looks like a huge sisters.

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Is ready to accelerate and to secreted in the environment, and this contain a lot of ganache inside there is some remnants of plasma membrane here, and this must be a huge secretory granule, and this is the reabsorption now of the the cup remains here and then gets reabsorbed like here. You see this may be the same like this, but it remains deep content and then the plasma membrane gets earlier reabsorbed, and here you see different stages.

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I think this is very important point in the life of embryo, because it has to make felt its presence in the room and to signal to send the molecules to signal to the woman start implantation.

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So that's it in the way and the this you see that the area over nine winners of mass is different, some micro life here- and this is the area where we believe that the implantation starts from a implants first and the behaves very aggressively.

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So this is I would like to thank my my wife for contributing also this German guys they did a fantastic machine allowing me to get very high resolution pictures and very long of course yeah. So that's all what I wanted to show and I saw a series of these blastocyst that you yeah. This is the top of the endometrium it adults, but I think it is a nonspecific attachment through dead cells.

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I, don't think it told us many things more than nice for the know yeah, so that's it I think I finished now photos messages that I can show you was around.

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This is interesting for me also to to ask questions from you to ask you questions about how to can I visualize all the embryo around with minimum of photos, because they take a long time. Okay,.

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I see you have a more slides there.

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It is this series of of the.

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Shoulders this.

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Is the Serie C is the same embryo in different aspects, and you need to put landmarks for John this one here.

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Okay, you will see it's here in the second photo. It's now here, the same thing this.

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Is my rotate in the middle right by.

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By rotating you can tell from from the middle also it is these two here, and this is the sister I'm sure before it's here and now the cyst is here- and this is here now we turn clockwise and the cyst is here- and this point this point from here gets to here here and then and another side here now the system this is the system and from Apple, so I turn it around as many times as two to help the whole thing.

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You know and that's why I put on the tip of the pin, so I have maximal area to be viewed because I cannot find any other method through my we were talking with dick that may be through. Magnetic forces can stay on the air, but this sounds difficult for the moment and for my notice of physics impossible.

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So that's it. Dick I have many words, but I didn't this because it will be redundant. You know, but in specific points is expandable sites.

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How much should this.

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So he can be published, for example, the new observations, the hatching no.

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Compaction and then I don't publish because you know now you can even to pay two bubbles. You know why should I pay? First.

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Baby somebody for this work myself, because I don't want to paint yeah yeah.

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I think it's not justice. This.

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Policy, you know, but I have the data sometime. Maybe I will publish yeah, that's already. What I'm doing now. It's sort of publication.

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Maybe finally, you.

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Know that they don't work this these glasses, they don't, they need special screens and software's that they work with polarized light.

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Have slides, but they are red.

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Blue, you know.

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It's primitive way, but it's the only way I can afford.

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I'm not sure where we.

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Everything sports, but you you need special software and special screens too, that they emit polarized light. You know it's expensive.

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To do this because, like this you've been given many colors, also well.

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They're, clear two tricks are available, don't require glasses, one is to present a stereo pair.

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It's a difficult skill, but if you, if you cross your eyes the right way, you can see it in three dimensions: yeah.

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The distance between the pictures has to be small enough, so it's the same as your distance between your pupils.

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The other way which you can do here with the needle is there is a psychological effect. Where is it? If you see a movie of a rotating object, then you see it in three dimensions as it. Never just movie stops I'm flat again yeah.

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He doesn't need help with something which we've been discussing. Marcos can rotate the embryos nice okay, there's a little bit of pewter. No because we're dealing with very small dimensions here and he, but it seems to me relatively simple software- to bring the images back to a common axis of rotation. So the thing to make a small gilded as they rotate, maybe.

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I can show now they say diesel food so but but I think would have given that it's you, let me find define justice open yeah now.

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See the other thing is: if we did, that should be possible. I know, there's some there's some literature on stereo microscopy from SEMS for producing a three-dimensional picture from the three dimensional data set from stereo views, these rotation all the way around, so it should be possible. We send this offer to make a pulse full 3d reconstruction of the surface.

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I think that would be an excellent place to start collaborating with your Kosan students to take these rotating. The embryos rotated to Tiffany pin start producing three-dimensional structures from.

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So, just to add I think you can actually do you could go to stereoscopic images. Then you can actually do it like a Google cardboard, so you can actually use a is the phone or something like that to actually see stereoscopic images, because you are basically projecting to their protein to images. The stethoscope of images and you see through a through a goggles cuff, using your.

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So you can actually do a very simple one. So there's.

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The Google glasses no.

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Kissing you're using your phone, your Android phone, you put it into a cat sort of structure, it's a car and then you put it.

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There was topic videos, yeah.

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There's a fellow his name's Richard Feldman. He had in fact I used to own one of those boxes, but it was larger and he he made stereo slides of model balance of all proteins and the.

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Allow you to superimpose the two images in stereo yeah.

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But now I think with Google I. Think he's got some API, so you can actually do it more easily. You can get you can do, I haven't done it, and but just because of the you know, accessory.

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I think how could I lenses, probably just prisms.

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Actually, it doesn't use a prism.

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Images and then you see.

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It's very easy to do.

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Software and I mean.

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In such a way that you can do it I think.

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Images into one image.

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360 degrees do I need to take all the angles like one by one or I can take some, and then a computer can put the rest and how many I guess that it will depend of the relief. Also of the surface did.

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And you know what I am muscle sure you can easily I mean at the moment. You can't I think you need to have a you need to create a thing to do it manually, it's very demeaning, mainly and, and you somehow have to use a software to to carve rotated, and you probably I, don't know how. How easy is to do the experiment and this year, because I think the electronic microscope takes a long time and basically you need to have a have a rec such that you can.

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You can rotate it yeah at the moment. You're rotating it by hand by the look of it and I think it is quite difficult. Then you need to basically that's what dick was saying that you need to find the corresponding points which you know what.

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Susan probably has a solution: Susan. Could it be possible to put a tiny motor inside the vacuum inside the SEM to rotate that Ken he's.

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Possibly there's any such machines as they do.

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Susan is working with very small motors to build this she's built a stage for rotating the axolotl embryos.

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What size that's the question this.

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Very small now.

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But helping the motor can you get inside your SEM yeah.

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Yeah you can put in see inside the museum and there are special motors like micro tools. I can I can show you right now on the web. There is a German.

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Hundreds of a degree the rotation each time, that's.

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It's very very I curated, no drifting, you know, but the course they're, not maybe five. Six thousand. Oh.

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Yeah, well, that's Susan how much it cost.

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I was wondering whether you can do it manually and you put a artificial marker somewhere, and then you hate it. Then. Therefore, you can use software to automatically translate or calculate how you know moves such that you don't need to bother.

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I, don't know, I, don't know I'm just guessing, so you basically have a specific point or a couple of coins that you can actually triangulate using software and let you know that it's there and then you manually, move around you manually, rotate it and then therefore you can, you may be able to reconstruct the whole thing in a movie.

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How many, how many I don't know I mean Thicke Maino, actually more but I mean normally, you need what thirty frames per second. So a lot of frames right and oh.

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You mean in terms of smooth rotation, yeah.

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Yeah, it's showing movies of people, it's about 30 frames a second, but it depends depends of course, on how fast you're rotating the I think. The thing is. The question really is the question of how much detail is lost. If you make the angle too big, he.

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Told me that he's doing there is a German guy doing it. He was the man of flight. He, his special stage is very expensive that they rotate they do all sort of of movements, and he told me that if the rotating object is rotates on the on its axis Sandra Sandra, then you can move one degree per frame. If it is eccentric the rotation, then you have to go much less in order to achieve a smooth rotation.

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Do you have your stage handy yeah? Are you sure, show everybody what you've got there, because this is makes small motors.

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Well, tell me: you can get smaller.

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You know you're off the edge Center, the camera.

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You go that's better.

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Now this this is a stage for holding the X law of embryo, and it has two different motors in it for two different notations and the whole course is what about $400.

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Be small enough to put inside the chamber and an SM.

C

D

It's cold I, bought it on Amazon.

D

Yeah, a high-tech.

D

A

Susan here here's your kosis I put it in the chat, your kosis contact information.

D

Yeah, oh thank you, respected, I, just ordered it on, like I, said that on Amazon.

A

Okay, so you're coach, you should be able to do it for maybe 100 hundred two hundred dollars, cuz you're just doing one rotation. What axis you you.

C

Know the hero wants to be to be compatible with the with a vacuum. You know and that's where you know what is combative.

D

C

See this yeah I save this game now and then yeah.

A

Well, that's that's overkill.

C

That must be like you say that yeah.

A

Well, that's that's ridiculous! It's too much general! This.

D

Looks very robust I would try it in a vacuum. Yeah sure.

C

Yeah to make lever to rotational tips, yeah.

A

You need to build it yourself, then it'll be cheap. Yes,.

C

This I asked for this and they told me 5,000.

C

A

All you tried to do is rotate a pin.

C

No I can rotate through the states here because five axis, but this is very enough for such a little guy mentioned you know, and if the jitters it yeah.

A

But that could all be corrected by software. That's not a problem. Yeah.

C

If this corrected by software, while spending so much time to do it perfectly, no because it is very time consuming for electron microscopy, one of these photos, maybe with two hours.

A

Let me ask you a question: how do you rotate a needle inside Bosnia, the.

C

Job that he did there are two two ways to add. A to one is: do I still the same spin I want to stop it because.

C

Stop saying will stop saying anyway. There are two ways of rotating I: don't know what the other you put perpendicular or horizontal horizontal is actually the tilt. The tilting is horizontal rotating. You know, because the tilting in this microscope works like this and they so yeah. There are buttons to do the self and some of our motorized, some, maybe not Bradley,.

A

Is the where, if you could you help them, get his screen share off, so we can that sure? What's going on.

C

Yeah yeah yeah I'm, fine.