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.
Vacuum Systems
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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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从本节课中
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
Different kinds of vacuum pumps have different ranges where they're effective.
And so to go from atmospheric pressure, to the ultra high vacuum
of a microscope column, you employ different kinds of pumps,
one after another, using the pump that's most effective in that pressure range.
The first pump that's commonly used is a mechanical or
rotary pump and the anatomy of, of this pump is that
the microscope column would be attached for instance, to this input tube.
And so gases from the microscope column as it's first being evacuated
would pass through here.
Now, within the mechanical rotary pump,
there's a rotor that's rotating around and around and around like this.
And attached to that there is what's call the Vane
with arms with a spring in the middle.
As it comes into a different position, the arms extend or contract
as necessary to meet the edges of the tube that they're found in.
And as a result, as this arm moves forward,
it pushes any gases that are present in this chamber,
and it pushes them along and it pushes them into the exhaust valve.
And from the exhaust valve they go through an oil mist trap and
are expelled into the room.
So the mechanical rotary pump just gradually scoops gases out of the column
and puts them into the room.
Now, mechanical pumps create vibrations that are easy to hear in the room.
And so, it's important not to be taking your very best highest
resolution images while the mechanical pump is on.
Instead, one waits until the mechanical pump is off, and
then you take your picture.
Mechanical pumps also require some maintenance.
You have to change the oil every once in awhile.
The mechanical pump can only reduce the pressure from atmospheric so
far, until the next pump takes over, which is the oil-diffusion pump.
The anatomy of this pump is that it the, it's usually mounted directly below
the electron microscope column, so the column connects into the top.
Gases from the microscope column diffuse into these chambers.
At the bottom of the pump, there's a reservoir of oil, and
that reservoir is heated.
And as molecules escape the surface of that reservoir,
they fly up and they hit these baffles, and
they are deflected by the baffles, and so they're bounce, they're bouncing down.
And the shape of the baf,
the baffles, cause the oil molecules to bounce downwards.
And when they hit these edges, these are water channels, with cooling water.
And so the edges of this oil-diffusion pump are maintained cold, so
as the hot oil hits the cold wall, it condenses here and
then runs down, and drips back down into the reservoir.
So the oil is constantly doing paths like this.
And as gas molecules from the column come in here,
they are hit by oil molecules and knocked down into the oil.
And pushed down further and further in through the pump.
And eventually they escape and are exhausted and
are passed to a holding tank that is pumped by the rotary pump.
Now, the oil-diffusion pump is quieter, but every once in a while at
the electron microscope you can hear a little pinging sound.
And that pinging sound can be oil molecules hitting the sides
of the oil-diffusion pump as they carry gas molecules down and away.
A very versatile pump that can actually start at atmospheric pressures, and
work down even to back an oil-diffusion pump is called a Turbo-molecular pump.
In the Turbo-molecular plump,
pump, it looks quite a bit like a series of three propellers.
Here's a propeller, here's a propeller, and here's a propeller.
And as gas molecules come down from the column,
they'll get inside between one of these propellers.
And as the propellers spin around and around, they bat the molecules down and
out, just like a propeller on an airplane.
And as the pressure is dropped lower and lower,
these vanes spin faster and faster.
The last stage of the evacuation is done by ion getter pumps.
And an ion getter pump is basically just two electrodes facing each other
with different charges.
The way these work is that if a water molecule from the column
enters into this chamber, every once in
a while there will be a cosmic ray that comes and
will split that water molecule into its component ions.
Once you have charged ions, they will be accelerated towards the electrodes.
So the hydroxyl ion will be accelerated and
actually embed itself into this positive electrode, and be captured there.
Meanwhile, its complement ion will be accelerated towards
the negatively charged electrode, and embed into that material.
Ion getter pumps can achieve extremely high vacuums.
But they work slowly, and they also can become exhausted.
Eventually, there can be so
much material embedded into the plates that they have to be changed.
This may happen once every three, four, five years.
One way to extend the lifetime of an ion getter pump, is to make sure that it only
is turned on when there's already a very high vacuum in the column.
If, for instance, you turn the ion getter pump on,
immediately after the column had been vented to atmosphere,
the ion getter pump would almost instantly become exhausted, and have to be changed.
The full vacuum system therefore is pretty complicated.
For instance in the column by the filament there's probably an ion,
ion getter pump evacuating the area just around the gun.
There are other ion getter pumps within the column.
At the bottom of the column, there's the oil-diffusion pump.
And that is backed, by the mechanical pump, with a storage tank,
a buffer tank, in between, and the mechanical pump exhausts to the room.
When a microscope is vented, first of all, all the pumps are shut off, and then
valves are opened to allow atmospheric pressure to vent into the column.
Once it's time to evacuate the column, the valves are sealed, obviously, and
then first, the mechanical pump is started.
And it evacuates the tank, the oil-diffusion pump, and
the column, as far as it can go.
Once it's achieved a strong vacuum in these regions,
then the oil-diffusion pump is turned on, and
it further evacuates the column, and pushing its exhaust into the tank.
As pressure builds up in the tank, once it reaches a certain threshold,
where the mechanical pump will, once again, be effective.
Sensors sense that pressure, the mechanical pump turns on,
it pumps down the tank and exhausts that into the room.
Meanwhile, the oil-diffusion pump continues to evacuate the column.
Eventually the pressure in the colium, column is low enough that the ion getter
pumps can be turned on, and they finish achieving the very high vacuum.
When a sample is inserted or film is exchanged or any other event,
however, that might perhaps lead to a burp of
air coming into the column, the ion getter pumps are all shut off, first of all.
And then the sample is inserted or
film is introduced in the column, and only after the vacuum
is re-established does, do the ion getter pumps turn back on.
Meanwhile the oil-diffusion pump is almost always pumping, and filling the tank.
And while you're doing your microscopy, every once in awhile,
it's possible that the mechanical pump will sense a high enough
pressure in the tank, and will turn on and evacuate the tank.
And for this reason, it can sometimes be advantageous before you start imaging,
manually cycle a mechanical pump.
Tell it to turn on and evacuate the tank to make sure that that tank is evacuated
and the mechanical pump won't turn on in the middle of your imaging.
