This class covers the fundamental principles underlying cryo-electron microscopy (cryo-EM) starting with the basic anatomy of electron microscopes, an introduction to Fourier transforms, and the principles of image formation. Building upon that foundation, the class then covers the sample preparation issues, data collection strategies, and basic image processing workflows for all 3 basic modalities of modern cryo-EM: tomography, single particle analysis, and 2-D crystallography. Philosophy: The course emphasizes concepts rather than mathematical details, taught through numerous drawings and example images. It is meant for anyone interested in the burgeoning fields of cryo-EM and 3-D EM, including cell biologists or molecular biologists without extensive training in mathematics or imaging physics and practicing electron microscopists who want to broaden their understanding of the field. The class is perfect as a primer for anyone who is about to be trained as a cryo-electron microscopist, or for anyone who needs an introduction to the field to be able to understand the literature or the talks and conversations they will hear at cryo-EM meetings. Pre-requisites: The recommended prerequisites are college-freshman-level math, physics, and biochemistry. Pace: There are 14.5 hours of lecture videos total separated into 40 individual “modules” lasting on average 20 minutes each. Each module has at the end a list of “concept check” questions you can use to test your knowledge of what was presented. As the modules are grouped into seven major subjects, one reasonable plan would be to go through one major subject each day. That would mean watching a couple hours of lecture and spending another hour or so thinking through the concept check questions each day for a week. Another reasonable plan would be to go through one module each day for a little over a month, or even three modules a week (Monday, Wednesday, and Friday) for a 3-month term. It is likely that as you then move on to actually begin using a cryo-EM or otherwise engage in the field, you will want to repeat certain modules.
Electron Guns
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来自 Caltech 的课程
冷冻电子显微镜（cryo-EM）入门
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Currents, Coils, Knobs and Names: Basic anatomy of the electron microscope
与讲师见面
Grant J. JensenProfessor of Biophysics and Biology; Investigator, Howard Hughes Medical Institute
Biology and Biological Engineering
To a first approximation, an electron gun is simply a bent wire.
So I'll draw a bent wire up at the top.
And through this bent wire, we pass a current.
[NOISE] And I'll draw electrons as green streaks, and so
[NOISE] as the current passes through this wire, a few electrons escaped the tip.
And so that they are firing out.
And the way to think about this process is it's somewhat analogous to water
flowing down a stream.
When water is flowing down a stream, every once in a while, there will be one water
molecule that collides with its neighbors in just such a way that it,
it receives enough energy.
That it can escape the surface of the liquid and
it evaporates out into the atmosphere.
In somewhat similar fashion, as electrons are streaming through this bent wire.
Every once in awhile, one of the electrons will collide with its in, its neighbors
in a, in, and the wire is warm because of the current flowing through it.
And sometimes an electron will get enough energy to escape the surface of the wire.
And metals have what's called a work function.
And then if an electron gets enough energy to escape the work function,
then it will fly out of the metal and
come down into the vacuum around it in the electron microscope.
And electrons fly out in
many different directions with many different energies as they come out.
And in an electron microscope, the wire itself is held at a very low voltage.
So for instance, in the bigger microscope at Caltech that we use frequently,
the wire is held at minus 300,000 volts.
And at the bottom of the microscope, there is a surface that is essentially grounded.
In fact, the bulk of the microscope is actually at ground.
So we'll go ahead and write that 0 volts.
And so because of that, there's a strong electric field between
the tip and the plate at the bottom that causes
the electrons to accelerate downwards into the microscope.
So that's the basic process.
The electrons are going to be accelerated down.
Okay, now let's undo those and draw the next element.
To help to focus this electron spray coming out of the gun,
there is an element that's often called the Wehnelt cylinder.
And it looks like a cup with a hole in the middle at the bottom.
And this cup is held at a voltage that's even more negative than the filament.
And so I'm going to write minus 300 kilovolt minus delta.
Another many volts, lower than the gun itself.
And because of that, the electric field lines in the vicinity of this,
of the hole in the cup [NOISE], they look like this.
[NOISE] And they serve to begin to focus the electron
spray coming out of the gun more towards a a central point.
And so the Wehnelt cylinder focuses the initial spray.
Now, the next element that the beam encounters is instead of going
all the way from minus 300 kilovolts to ground in one step,
there are, in mic, in electron microscopes what are called accelerator stacks.
And the accelerator stack is just a series of disks with holes in them.
And I'm going to try to draw ten here.
So these are, these look like washers from above.
But here in cross section, we'll draw them as lines with a gap in-between.
Nine of them and a tenth one.
And at this point, we can go ahead and erase that.
And this accelerator stack has, is connected by a series of resistors.
Between each of the plates in the accelerator stack.
And the lowest plate is grounded,
as is the rest of the microscope.
So, this plate is grounded.
It's at 0 volts.
And because of the resistors in between each of the plates,
there is a graded drop in the potential.
For instance, this plate might be at minus 30,000 volts.
And this one, minus 60,000 volts.
This one, minus 90, minus 120,
minus 150, 180, 210, 240, and
the top plate might be at minus 270,000 volts.
And because of this graded potential,
the electric field lines in this region are flat,
very nearly flat, and perpendicular to the beam.
[NOISE] And of course,
these field lines continue all the way down between these plates.
I won't draw them all.
And because of this, as electrons enter this zone,
they come to it and they accelerate through the accelerator stack.
And by the time they emerge, they have gained their,
they've gained 300,000 volts.
Now, the speed of electrons that have gained 300,000 volts
turns out to be 76% of the speed of light.
So the electrons are moving at nearly the speed of light.
Their wavelength is 2 picometers, which is just 200ths of an angstrom.
So clearly, the wavelength of the radiation
being used in an electron microscope will not be the limiting factor.
The wavelength is small enough that you could see
details much smaller than a single atom.
Now, this is the basic design of an electron gun.
There's a few other things that I would like to mention about it.
The first is that the whole system is contained in a very high vacuum.
And that's because electrons have high-scattering cross-sections
with material.
And so if there were any other gases in the chamber,
many of the electrons would scatter from those gases.
And we would like them to pass unscattered,
from the gun all the way to our sample, and then through the lenses.
And so, the chamber has to be at very high vacuum.
Nevertheless, there always is some water vapor and
other gases that do get into the column.
And sometimes, these contaminants will build up
stalactite-like deposits on this, on these accelerator stacks.
And the stalactites every once in a while will get tall enough and
the voltage gap between them will be sufficient that there will be
discharges between these stalactites.
That break the shape of the electric field and
they can also damage the electronics in the instrument.
And so, in order to clean out the gun occasionally, guns will be conditioned.
The word is conditioning, and all is meant by conditioning is that the total
voltage of the system is raised even higher than the normal operating voltage.
So for instance, if we want to operate our microscope at 300,000 volts,
we may condition it at 315,000 volts, for instance.
So that any discharge that's going to happen occurs.
And then we can lower the voltage and reach a more stable working condition.
The last thing that I'd like to mention about electron guns is that
in order to sustain voltage gaps of 300,000 volts or more,
there is in the the, the tank that creates this high voltage.
The high-voltage tank that is insulated with a gas
sulfur hexafluoride gas, which is a good insulator and
helpful for the high-voltage tank.
But it's, it's somewhat dangerous in that sulphur hexafluoride
is an odorless gas that's heavier than oxygen.
And so in any electron microscope room you'll probably find a tank of
pressurized sulphur hexafluoride.
And if the high-voltage tank or that tank of compressed gas developed some kind of
leak, you can have sulphur hexafluoride coming into the room.
And you won't smell it, you don't see it.
And because it's te, heavier than oxygen, it'll settle to the bottom of the floor.
And in a disaster scenario, if someone were to hit their head and
fall on the floor, or maybe go to sleep on the floor, or for any reason their head
was in the zone that was full of sulfur hexafluoride, they could be asphyxiated.
And so many microscope rooms,
you'll see with vents on the very bottom of the floor.
And they maybe have sensors, oxygen sensors to that,
that will go out, off if there's a lack of oxygen.
So just to give you an idea of what some of this looks like, this is a picture of
the accelerator stack from our Polara 300 KB microscope that we use at Caltech.
This is when they were replacing the filament.
And this is the box that contains the filament itself.
And then this is the accelerator stack.
If you see this stack of rings here,
there's about ten of them, that's the accelerator stack inside.
And it has red lights on it because they're heating it up,
trying to de-gas the materials before they enclose it.
And then begin to pull the vacuum in the vicinity.
And here, as I mentioned, the bottom of the microscope is grounded.
And it's interesting that it's really grounded well.
It's important to have a very stable microscope electronically.
So for instance, when we grounded when we prepared a ground,
for our microscope at Caltech, this is what it looks like.
Actually, the hole is about 12 feet deep with a huge copper pipe going down it.
And these are the grounding cables about an inch thick that connect to it.
So it is solidly connected to ground.
So at that introduction to electron guns,
now we need to talk about the concept of coherence.
Now, there's two types of coherence that we need to think about.
The first is called spatial coherence.
And what it means in the context of electron microscopy is,
do all the electrons come from the exact same direction?
Now clearly, in an electron microscope, if some of the electrons are coming straight
down through the sample to hit the camera at the bottom.
And then another electron comes from a different direction and
goes through the sample in a different way.
It's going to hit the camera over here.
And so, each electron produce an image of the sample in a different place.
This electron produces the image right there.
And another electron might produce an image over here.
And if those images are added up, you get a blurry image.
And that is poor spatial coherence.
So one of the keys of an electron gun is that all the electrons need to come out of
exactly the same position in the tip.
The next kind of coherence is called temporal coherence.
And in this context, it means, do all the electrons have exactly the same speed?
The reason this matters, is imagine if one electron comes at 300,000 kilovolts,
it has a wavelength.
And because of that certain wavelength,
it will be focused by the lenses in the microscope.
And it'll be focused at a certain position.
And that's where we'll put our camera.
But imagine if the next electron comes with 301,000 volts.
Then it's going to be moving more quickly.
And because of that, if will focus less strongly in the lenses.
And so it may focus at a lower plane.
And so if we build up an image from electrons with different speeds,
we'll have an image we'll have to put our camera somewhere.
And that camera will be receiving images from each electron that are more or
less in focus.
Some are in focus above it, some are in focus below it, and
the result again is a blurry image.
And so one of the keys to an electron gun is that all of the electrons come out of
the tip with exactly the same energy.
And that's called temporal coherence.
Now, this leads us to understand the three
most common types of filaments used in electron microscopes.
The first is a tungsten filament, and
in it, it's really just a bent tungsten wire.
And tungsten filaments are fairly cheap, they're quick to replace,
and they're common in many, many microscopes.
But they're not very coherent.
The next kind of tip that you'll find in electron microscopes is called
a LaB six filament, which is a lanthanum hexaboride crystal.
And in this case, the gun itself is a small crystal of lanthanum hexaboride.
And because the crystal has a very sharp tip,
the electrons will come out more nearly in the exact same position.
And they'll have more nearly the same energy as they escape
that crystal lattice.
Finally, there are field emission guns.
Now, a field emission gun also has a very sharp polished tip.
The reason it's called a field emission gun is because below the tip
a, an electric field is used to actually pull on the electrons in that tip.
And so they're pulled out of the tip.
And because of that, they come out with much greater coherence.
And the way to think about this is to think about, say, a volcano.
And a volcano as it erupts, you have pieces of lava that come out in
all possible directions and with all different speeds.
And some fly really far, some fly relatively short distances.
This is poor coherence, both temporally, how fast they come out.
And spatially, because they come out in all directions.
That's like a tungsten filament.
On the other hand, a field emission gun is more like a child's slide.
Now on a child's slide, imagine a slide and a whole bunch of balls at the top.
And each ball is positioned just at the edge of the slide.
And if you tap the ball, it will just start to roll down the slide,
and it emerges down at the bottom of the slide.
And every single ball that you do that to will come down and
end up going exactly the same direction with almost exactly the same speed.
Which is the the kinetic energy that it got by converting the potential energy
falling down the slide into kinetic energy.
This is like the field emission gun.
By having electrons in the tip, not quite escaping but
then pulling on them with a field.
As soon as they emerge, then they're accelerated through the accelerator stack
and they come through from exactly the same position,
in the same direction, with the same speed.
And so field emission guns are the most coherent.
Now, there's two types.
There are what's called warm field emission guns,
or thermally assisted, and there's cold field emission guns.
And the difference is that in a cold FEG, or field emission gun,
the electrons are drawn out of the gun entirely by the field.
In a thermally assisted field emission gun,
there's a substantial current going through the tip.
And that current heats up the wire so that the electrons are closer to
having the energy they need to escape the wire and be extracted.
So field emission guns, however, are much more expensive and
they may take days to replace when their lifetime is over.
Because these filaments require heating, they degrade over time.
And it's important to set the temperature of the filament optimally.
The warmer the filament is, the more electrons will come out.
And so you have a brighter beam but,
at the same time, sometimes the, the metal of the wire will actually degrade.
And the quality of the tip, the fineness of the tip, the sharpness of the tip
will degrade over time and eventually they have to be replaced.
And so when you're taught exactly how to use the electron microscope,
it will be an issue of how to find that right temperature where
you warm up the filament tip enough to get the brightness you need.
But no more, because then you will shorten the lifetime of your tip.
