Placeholder Image

Subtitles section Play video

  • Hi, my name is Eva Nogales, I'm a professor of molecular and 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 and 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 matter,

  • how the electron microscope is able to generate

  • contrast in visualizing sample

  • and then give you examples of biological molecules and cells

  • and how the images are generated, how they look and

  • how we go into farther processing to learn more

  • about 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 scanning electron microscopy, is a method that uses

  • focused 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.

  • It's a crab larvae and it looks very pretty because it has added false color,

  • but 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

  • Let me give you another one, this is of dust mites,

  • these are nasty things that cause allergies certainly they do only.

  • And I want you to pay attention to the scale bar here,

  • which is two hundred microns, this is about the size of these organisms.

  • You could 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 these kind of tissue has lots of cells that have these cilia that they use to create fluid motion

  • and allow the sperm and the egg to encounter one another.

  • Notice that the scale bar now is 2 microns, so we're really seeing cellular details right now.

  • I would like to concentrate from now on on the other electron microscopy technique,

  • which is Transmission Electron Microscopy, or TEM.

  • This uses very different principles and what it gives you is a projection

  • images at much higher resolution of thin samples that are introduced into the microscope and there's possibility here

  • to get even to an atomic resolution,

  • as I will tell you in a second.

  • So this particular example comes from a section that was taken from the flagella of a sperm,

  • the one that could have been coming up the oviduct that I showed you before.

  • So, in this section, what we see is the axoneme,

  • a structure that goes all the way along the flagella,

  • it's made up by microtubules, which are organized into this beautiful structure.

  • Now, microtubules are made by self-assembly of the protein tubulin.

  • and you can actually purify tubulin, typically from mammalian 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 contains information at the atomic level.

  • The scale bar in the image that I showed you before

  • was not microns, it was nanometers.

  • And what you see here, the thickness of one of these microtubules is 250 angstroms, or 25 nanometers.

  • In fact, this methodology, has been used to obtain information about the structure of the tubulin protein itself.

  • And what I show you here is a density map in blue inside the protein molecule, where these yellow lines represent the polypeptide chain

  • going along it. And every one of these squares in blue corresponds now to one angstrom.

  • So the resolution here is much higher, is at the atomic level.

  • So, I want 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 matter.

  • In the transmission electron microscope, electrons have very high energies,

  • of the order of hundreds of kiloelectron volts.

  • That means that they are moving at close to relativistic speed,

  • They are moving so fast that sometimes they basically go through matter 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 going to go through and bounce off 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 speed,

  • but change direction. And this is what we call the good scattering

  • and is going to be used to generate contrast in the image, in the TEM.

  • Unfortunately, there's another process of scattering,

  • the bad scattering, and these are electrons in your primary beam generated in the microscope

  • that go through the atom and interact with the electrons in your sample

  • and in doing so, they lose energy of their own and they pass it to your sample.

  • These inelastically scattered electrons

  • are going to only contribute to the noise in your image,

  • while at the same time, really damaging your sample.

  • Ok, so, the TEM is going to be able to visualize your sample

  • by using these elastically scattered electrons to generate contrast in the image.

  • And the way this is done is in two different manners.

  • 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 maize 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 doctor's, where more X-rays are being absorbed by your bones

  • than by the rest of your soft tissue,

  • so the bones appear darker in the image.

  • 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.

  • Ok, 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 by means of an aperture.

  • The aperture is placed after the objective lens

  • and what is 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 happens, because there's more density, appear dark

  • and regions where there was less scattering appear bright.

  • 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. This is the contrast

  • that is generated in these types 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 unscattered 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 matter, the wave front will suffer a phase shift

  • and this is what will ultimately cause the interference, which will mix with the unscattered wave front.

  • I'm not going to into any more physics, rather, I will now show you how an electron microscope looks like.

  • Ok, this is what I would call a middle of the range electron microscope.

  • 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 vacuum

  • 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 of the sample, how bright and how large is the area that's being illuminated

  • and then comes the most important lens in the scope,

  • which is right in the middle. It's 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 phospo screen, directly on a TV

  • and ultimately recorded for further data processing,

  • either on photographic film or on a CCD camera.

  • The one thing that I would like to show you is an electron microscope grid.

  • It's right there on my finger. This metal grid is coated by a thin layer of carbon

  • and then the sample goes in there.

  • And we need extremely little amount of sample.

  • We use, say for purified protein complexes, concentrations that are on the nanomolar,

  • sub-micromolar range and we use a very small drop, the size of small tear,

  • that goes right in there. And then gets blotted to a thin layer

  • before it's very quickly frozen.

  • The grid, all of this is done, under liquid nitrogen or nitrogen gas and then in the holder

  • and then it is introduced in the electron microscope.

  • But with very, very little sample, we can still get millions of occurrence of the complex

  • we are interested in and take many, many images and hopefully get the structure we need.

  • I get often asked what kind of resolution can the TEM reach.

  • And in fact, microscopes like the one that I showed you is capable of obtaining images

  • with atomic detail. This is such an example,

  • this corresponds to a thin, nanocrystal of silica,

  • where each one of these dots are actually atoms that have been visualized in this image.

  • In fact, a state of the art electron microscope can reach resolutions

  • beyond a single angstrom. 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 angstrom.

  • 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 reached

  • 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 is 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 samples.

  • 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.

  • When inelastic scattering 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.

  • Ok, so, how do we overcome these problems?

  • There's two solutions. The first one to occur historically was negative staining.

  • In this case, your sample is embedded in a low concentration of a salt solution

  • of a very heavy atom, typically uranium.

  • The sample is embedded 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, the uranium, generates very high contrast

  • so we can overcome 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 of radiation damage.

  • The protein may vaporize, but as long as the cast still reproduces its shape,

  • we are ok. So, the big pluses of this methodology are the high contrast in the image,

  • and the fact that it's fast and easy.

  • So, Berkeley undergraduate students 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 minus, the big minus, is that artifacts are readily 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 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 that's the ultimate size of anything that you're going to be able to see,

  • which is typically about 15 angstroms.

  • So, the solution, the second 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 active solution where it is happy,

  • but then is very quickly frozen. It's frozen so fast that the water molecules

  • don't have time to reorganize into a crystal

  • into ice, and therefore remains amorphous. We call that vitrified water.

  • 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 altogether.

  • 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 other means,

  • I'll tell you about that later.

  • Radiation sensitivity is limited because now the very low temperatures means 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 set-up,

  • you can even obtain time resolution.

  • You can time a certain biological process to be triggered during the process of vitrification and trap 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 cryo-EM.

  • It takes many months, if not years, to really master.

  • The other problem is the contrast, as I told you.

  • There are ways of enhancing the contrast in the electron microscope,

  • but they always tend to come at a price.

  • So, we mostly we deal with that computationally.

  • The other problem is that although we have minimized radiation,

  • still the sample remains sensitive. So, we have to use

  • low doses, typically 10-20 electrons per angstrom squared.

  • And that means that the images are going to be very noisy.

  • So, let me give you an example how the same sample. the same biological material

  • looks like in negative stain versus cryo-EM.

  • 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