Lights, lasers and microscopes
Microscopes can be used to see finer and finer details of biological specimens such as T-cells. These are about 10 microns in size - which is about 10 times smaller than the width of a human hair. Ed Sanders from the Lee Lab at the University of Cambridge walked Anoushka Handa through these imaging techniques...
Julia - I can't believe you can print a microscope, but surely we need other instruments to let us really see the details of say, our own cells.
Anoushka - Well, there are different types of microscopes that let us see finer and finer details of small specimens like human cells.
Julia - How big was that immune cell? You said you were supposedly standing in?
Anoushka - That immune cell called a T-cell was about 10 microns in size. Now that's about 10 times smaller than the width of a human hair. And the proteins on that cell are even smaller than that.
Julia - So how do you look at them?
Anoushka - Well, using light lasers, tags, and electrons to zoom in further and further on these pretty small cells, Ed Sanders from the Lee Lab at the university of Cambridge, walked me through these imaging techniques.
Julia - Anoushka you didn't tell me we were gonna play laser tag.
Anoushka - I've headed over to the Department of Chemistry to have a look at a human T-cell, under a microscope. T-cells are a set of white blood cells that are part of the immune system. They're activated when fighting infections and foreign particles. You can imagine them as a bodyguard of sorts, which comes out when those cold or COVID particles come and attack.
Ed - It's quite important to know how T-cells make these decisions and how the decisions downstream would lead to either someone fighting off a disease or in the case of vaccines, how the vaccine would work is all due to your T-cells recognising something and then priming itself for a later infection. We know that T-cell triggering would lead to the immune response. Those very initial stages are actually very poorly understood. And partially this is because of the lack of tools to look at them properly.
Anoushka - Now, there are certain tools that we can use to help us understand the initial stages, and microscopy bears a large burden of this. There are various kinds of microscopy, electron microscopy and fluorescence microscopy, but how do they work? And which would be best to use in this case?
Ed - Electron microscopy would look at a difference in electron density. Whereas fluorescence microscopy would look at a molecule tagged with something that glows and you can use the glow to say where things are. So we could look at how certain molecules move on a T-cell surface with fluorescence microscopy. Whereas you could not do that with electron microscopy.
Anoushka - We're gonna be having a look at some human T cells.
Ed - First we're gonna use normal white light microscopy. So this is what you'd see in a typical biology lab in schools. And essentially what you'll see is a spherical blob.
Anoushka - Is that a technical term?
Ed - No, it's not a technical term at all, but it's the best way to describe what you're going to see.
Anoushka - Wow, that's incredible. These are human T-cells that I can see under the microscope. And what I can see is like Ed said these spherical blobs, but I can also see these little lines coming out of these spherical blobs. What are they?
Ed - Those are microvilli. These are long fingers that the T-cell will use to help it scan for a pathogen.
Anoushka - So these fingers I can faintly see them. Can we just use electron microscopy to have a look at the structure of them?
Ed - Well, electron microscopy would do that very well, but they can't necessarily look at the single molecules that are involved when the T-cell would make a decision of, whether something is a foreign invader or whether it's something that's meant to be there in your body.
Anoushka - Now, this is where a type of fluorescence microscopy comes in called super resolution microscopy. Super resolution microscopy is a type of fluorescence microscopy technique. You sacrifice temporal information, time for spatial information, space. You can have a look at anything to be honest. So long as you can tag it with a fluorescent dye, we're able to stick it on our microscope, illuminate it with our laser, and a subset of these fluorescent dyes are turned on at different points in time. And by doing so, we create this image of a bunch of different points, which we can then collate together and get our data from. And that way, not only are you passing the diffraction limit of light, which is 250 nanometers, you're gaining information that's already there, just couldn't be seen in normal fluorescence microscopy. But what processes happen on this scale?
Ed - The T-cell example is actually one of those processes, a single T-cell receptor and the interaction between that and an antigen can lead to downstream signaling. So we need to be able to get down to single molecule scales, where we can actually see what's happening on that level.
Anoushka - How do you stop fluorescent molecules from turning on at the same time?
Ed - So there's a few ways to do this. You can either use molecules that are photoactivatable, so we'll only turn on when exposed to UV light. You can use chemistry to help the molecules turn on and off. Or you can have a probe in solution that will bind and unbind. These three techniques are essentially the painter's palette for super resolution microscopy.
Anoushka - You've kindly labeled a T-cell with a fluorescent molecule. And what are we actually gonna look at when you image?
Ed - So today we're actually going to look at the membrane of the T-cell. These fingers actually play quite a big role in T-cell signaling. So we need to be able to accurately identify the structures. When I turn the lasers on, what you'll see is you'll see little flashes of light on the camera. So spots and those spots are single molecules in your sample, turning on. That's literally a single molecule that you're seeing there glow.
Anoushka - Incredible. We're seeing this in real time. How long does it take to actually get an image or a structure of the cell?
Ed - It'll take hours, because every time that we look at a single molecule and decide where that is, essentially, we need to build up the full picture of the T-cell. We need lots of molecules to turn on and build up this pointillist image.
Anoushka - Can we look at any other proteins on the cell at the same time?
Ed - For sure. So with the cell membrane, what I've done is I've used a probe that will bind the cell membrane in and then leave and bind on and off. If you wanted to look at a protein, you can use an antibody to bind that protein, and again, you'd have your fluorescent molecule there. All you really need for super-res is a way to attach a fluorescent molecule to a target of interest. And then secondly, somewhere to make those molecules blink.