OK so the title is hyperbole, but I’ve definitely struggled recently with my cryo-electron microscopy. I guess here I’ll give an overview of what exactly electron microscopy is and why I’ve struggled.

Professor Jensen of CalTech has a great series of videos on Cryo-EM. Why we use it, how we use it, and what it is. Anyone interested in the technology should watch it, but for my own purposes:

• Cryo-electron microscopy consists of freezing a sample and then shooting electrons at it to see the 3d structure of it at the smallest atomic scales.
• We’re using it to study a number of proteins that cause diseases. In particular we want to know how the 3d shape of a certain protein creates that protein’s function. And how that function can then go on to cause a disease.
• So we purify a specific protein, make a cryo-grid from that purified protein, and then look at that cryo-grid under electron microscopy hoping to get a good 3d structure.

But that’s where the problems start. First of all, purifying a protein to 99.9% purity is no small feat, especially when you’re taking proteins out of actual patient samples. I’ve dearly struggled to get the required purity that would be needed to make good grids for imaging.

But once I have some “pure” protein, I need to add it to a grid to image it. A cryo-grid is a 1 millimeter by 1 millimeter circle about 1 micrometer thick. On that grid are cut out many 1 micrometer by 1 micrometer squares. And in each square are a mesh of 100 nanometer by 100 nanometer holes. When I add a tiny drop of my protein sample (which is in water) onto the grid, the hope is that the proteins will settle down into the holes. I will then “blot” the sample by pressing some paper onto both sides of the sample, which wicks away all the water not in the holes. I then instantly plunge the sample into liquid ethane, freezing all the liquid in the holes in an instant.

What you get is supposed to be a grid covered in a tiny thin layer of ice, and in each hole the ice contains your proteins of interest. Since they were flash frozen in ethane, the ice here is “vitreous,” which means glass-like. It’s see-through just like glass. And so a beam of electrons can pass into the ice to create an image of the proteins inside the ice.

But there’s problems. Let’s get back to making the grid: most proteins are hydrophilic which means water-loving. The opposite of hydrophilic is hydrophobic which mean water hating, like oil. Oil and water don’t mix, and neither do hydrophobic and hydrophilic things. Our grids are made of copper covered in a layer of carbon, and that stuff is naturally hydrophobic, meaning it doesn’t interact well with the hydrophilic proteins (and the water they are in).

So before adding proteins we have to glow discharge our grids. This means putting them in a machine that shoots broken-up water molecules at them. Those broken-up water molecules have oxygen in them, and some of them will bind to the grid creating oxygen-containing compounds. Those compounds are very hydrophilic, so the whole grid becomes hydrophilic enough for the proteins to interact with it.

At some point we got a new glow discharger, and I swear that it started destroying my grids. Like I said the grids are tiny and fragile, 1 millimeter across, 1 micrometer thick! This glow discharger shoots water at them, and the new one shot the water so hard that it was punching through my grids and destroying them completely at the microscopic level. I couldn’t see the damage because it’s microscopic, but after adding the protein to my grids and flash-freezing them, I’d look at them under a microscope and see nothing but a completely destroyed grid. I finally just stopped trusting it completely and moved on to using a new glow discharger that’s a bit weaker.

So OK I solved the glow discharge problem, but now here comes the ice problem. Like I said above, you want the proteins to be encased in glass-like vitreous ice. If you have no ice, well you have no proteins. And if the ice is too thick, it’s no longer glass-like and you can’t see through it. I kept being on both sides of those extremes, first I had ice so thick I couldn’t see anything, then I had no ice at all. You are supposed to manage this problem by configuring your blotting time, which is how long you wick away the water before plunging the grid into the liquid ethane. Shorter blot time, thicker ice, longer blot time, thinner ice or no ice at all. Try long and short times to get the ice just right.

And yet I was using ultra-short blot times and still getting thick and thin ice sometimes at random. On the balance I got more grids with no ice at all, so I kept thinking I needed to drop the blot time more and more. My adviser said that there is a minimum blot time of about 2 seconds and you never want to go lower than that, but I tried 2 seconds and the ice was still way to thin or non-existence. That seems to say that my blot time is still too long, yet 2 seconds is as short as I can go.

I finally asked an expert in the chemistry department who suggested I used their facilities instead. He also suggested that 1 second of blot time is perfectly fine, and so that was what I did. I FINALLY seemed to start getting good grids, so let’s hope it hold out.

So I’ve struggled with glow discharging, and then blot times, as well as protein purity. I’ve finally got some good grids, and I hope I can collect a lot of data on them. If I do that, I may be able to get 3d structural information using AI and a whole bunch of analysis. We’ll see though, we’ll see.