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.
Sample Prep Grids
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Del curso dictado por Caltech
Getting started in cryo-EM
211 calificaciones
De la lección
Fundamental Challenges in Biological EM
Conoce a los instructores
Grant J. JensenProfessor of Biophysics and Biology; Investigator, Howard Hughes Medical Institute
Biology and Biological Engineering
Obviously, one of the most important aspects of sample preparation
is the choice of which EM grid, which kind of EM grid, and
what kind of a surface, is used to support the sample.
Now, there are lots and lots of different kinds of grids.
They're all three millimeters across, and very thin obviously.
But they have different patterns of the support structure.
One class of grids are characterized by a mesh, and these have
grid bars across the grid, both horizontally and
vertically, and the mesh refers to how many grid bars there are per inch.
So, for instance, a 200 mesh grid has spacings like this.
A 300 mesh is more bars per inch, so the bars are closer together.
And you can even get 400 mesh grid bars.
Another kind of grid is called a slot grid.
And here there's just a very large slot in the middle that's left completely open.
Another kind of grid is called a finder grid.
And the idea of a finder grid is that there's a number of symbols like this
asymmetric object in the middle, and this large arrow here, and
then the numbers that are actually imprinted and the letters.
A finder grid helps you identify a particular grid square that,
of interest and maybe return to it later or label it because all of
these landmarks that are placed around it allow you to identify a particular square.
These are particularly helpful for clem experiments for instance.
Now the grids are commonly made of copper, but
you can also buy grids made of nickel, or gold.
The advantage of gold is that it's not at all toxic to cells.
And so if you want to grow cells on a grid for
days in a row, it's gold is a good choice because it's an inert metal.
And sometimes people use grids made of molybdenum for
reasons that we'll describe later.
Now for some samples, it is possible to simply apply the sample to the naked
grid itself because a sample will span all the way across the grid bars.
However, it's much more common to apply some kind of a coating
on top of those grid bars.
One common coding is formvar which is a form of plastic.
Sometimes on top of the formvar is deposited a thin layer of carbon.
Another popular surface is holey carbon.
And so here's a picture of holey carbon.
Just meaning that there is a carbon sub-straight here
at this reticulate network of carbon
with lots of holes in between that can provide nice regions to image.
Another popular grid surface is called quantifoil.
These grids are made by a process that allows both the size of the holes and
their pattern to be very carefully controlled.
And so you can order a grid surface with any size hole you like
in whatever pattern you like.
And finally, one might take a grid with holes in it,
either the holey carbon film or perhaps a quantifoil grid and
cover it with another thin layer of continuous carbon.
In that case a cross-section through the grid might look like this,
where these are the heavy thick grid bars of copper.
And on top of that is a layer of carbon with holes in it, and so
in cross-section it might look like this.
And then on top of that might be deposited yet another layer of very thin carbon,
perhaps just five nanometers or so.
Another thin layer of carbon on top of that.
Followed by the sample of interest with say individual proteins
in suspension in that fluid.
Now the way these very thin layers are produced is a process called evaporation.
For instance carbon evaporating.
The way it's done is imagine a chamber here, and
theres a platform on that chamber on which a grid is placed.
And then, above that is a thin rod of let's
say carbon, a sharpened carbon rod.
And the chamber is evacuated.
So there's a vacuum inside and then a current is passed
through this rod causing it to heat up.
And as the rod heats up tiny little
clusters of carbon atoms will come raining down away from that tip.
I suppose they rain in all directions, but the ones of interest
are the ones that rain down and cover our grid with a very thin layer.
And depending on how long you apply the current, the,
the layers that's deposited gets thicker and thicker.
Now it has been observed that as soon as a layer of carbon is deposited through this
carbon evaporation, it's quite hydrophilic.
And so if you apply a drop of fluid that fluid will just immediately wick all
the way to the edges of the grid and behave well for sample preparation.
However, if those grids are, stored for weeks in a row.
The surface of the grid often changes somewhat, and they become hydrophobic.
And so, if you applied a drop of sample on the grid later
you would see it bowl up, because the grid itself was so
hydrophobic, and this can make getting good grids very challenging.
And so, it's common practice before grids are used to do what's called
glow-discharging to restore their hydrophilicity.
Glow-discharging is done in a device, very much like a carbon evaporator, in fact,
some are dual purpose devices, but imagine in a glow discharger.
Again, there's a platform upon which your grid rest.
And the grid is covered with a layer of carbon.
But this time, instead of a rod being evaporated, there's a plate here and
a plate here and a big voltage difference between those two plates.
And again a vacuum is pulled in this chamber.
Because the vacuum isn't actually perfect you'll have still a number of water
molecules from the atmosphere that was in the chamber as the situation was set up.
And there's always ionizing events that happen.
You know cosmic rays can come into this chamber and cut one of these bonds,
resulting in a hydroxyl ion and a proton being liberated.
And because of the voltage gradient between these two plates,
the proton might be accelerated towards the upper plate,
while the hydroxyl ion is accelerated down towards the lower plate.
And so you have a situation where the carbons surface
is hit with a barrage of charged ions.
And as these charged ions hit the surface, it's thought that they produce alcohols
and carboxylic acid groups as well as aldehydes, ketones, and esters.
And perhaps all kinds of different species that are now hydrophilic.
And so, after glow discharging it's seen that the grade,
the grids behave much better, much like after they were recently made.
For certain samples, it's been seen to be advantageous to,
after the vacuum is pulled, to leak in
through a valve here attached to some kind of vial.
Some other substance, for instance, polyamines have been used.
And so when you have a, a, s, a low concentration of polyamines here,
that are ionized and accelerated towards the grid.
That can give the grid a subtly different surface chemistry.
Finally, another variant of this device is called a plasma cleaner.
And plasma cleaning has the same overall goals.
Now unfortunately, if you have a grid or the grid bars are made of say,
copper, so, here, for instance, is a grid bar, here's a gird bar, here's a grid bar.
Grid bars are made of copper as is the entire outside of the grid as well.
But you have a thin layer of carbon over those grid bars.
And so here is the thin layer of carbon between the grid bars.
And you plunge freeze such a grid.
Unfortunately, carbon and
copper have very different coefficients of thermal contraction.
And so as they rapidly cool down, the carbon contracts less than the copper.
And the result has been called cryo-crinkling.
And it's seen here, where the size of each grid square
is now made quite a bit smaller than the carbon surface that originally covered it.
And so the carbon ends up crinkling and rippling.
And this can be a severe disadvantage for
say, tooty crystallography as we'll see later.
And this is why malibdomin grids have been used
before because the coefficients of thermal expansion contraction for
malibdomin and carbon are much closer to the same.
Most recently, Laurie Passmore has developed methods to create
the entire grid out of gold, both the bars and also the surface
layer with the holes in it, so that it has exactly the same coefficient of shrinking.
It has been found that this reduces beam due specimen movement.
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