Name: Biotechniques: Behind the Technique. Super Resolution Miscroscopy with Steven Lee
Description: Biotechniques: Behind the Technique. Super Resolution Miscroscopy with Steven Lee
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Duration: T7M07S
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Content URL: cambridge.mp4
Upload Date: 2018-11-08T13:01:00.8330000
Transcript: Language: EN.
Segment:0 .
Segment:1 What sparked your interest in this field of research?.
STEVEN LEE: We're interested in developing biophysical techniques to address biological problems. And as a background, my background is in physical science. I'm a chemist. And what strikes me in the field of biology is there's lots of really important questions for which there isn't technology to be able to address it. So in general, we're interested in just kind of building instruments, building optical microscopes to be able to visualize biological processes.
STEVEN LEE: And I find it really incredible that we don't necessarily have those instruments to be able to see these things. And so we use a very specific kind of microscopy called super resolution microscopy, which looks below the diffraction limit. So if you imagine taking a magnifying glass, and just magnifying, and magnifying, and magnifying, at some point you can't see any more on that image. And there's some kind of innate blurriness to light.
STEVEN LEE: And that's called the diffraction limit. And so for optical wavelengths, so the light we see, that size is about 250 nanometers. Now, unfortunately, 250 nanometers is much larger than the spatial scale that a lot of biological processes occur on. And so there's been a huge effort in kind of physical scientists trying to develop optical microscopy to be able to see below the diffraction limit.
STEVEN LEE: And, in fact, this was awarded the Nobel Prize in chemistry in 2014 for being able to beat the diffraction limit, and actually to be able to see below that kind of innate blurriness of light to see what's going on, on a biological spatial scale.
Segment:2 How does super resolution microscopy work?.
We use fluorescence. So if you've not heard of fluorescence, it's a very simple phenomenon. It's just that when a fluorescent molecule absorbs light of one wavelength and spits out light of a slightly redshifted wavelength. A classic example, if you've ever been in a bar or a club and you've seen someone with a blue glowing drink, that's almost certainly gin and tonic because there's a compound in tonic water called quinine, which is fluorescent.
So what we do in our lab is we don't use quinine, but we use similar molecules. And we covalently chemically attach those fluorescent molecules to the biological molecule we want to see. And then what we do is we use lasers. We excite the fluorophore with a laser light, and then collect that using very sensitive optics. So we have very sensitive cameras, which typically can detect single photons.
So they're extremely sensitive to very low levels of light. And then we use that information to infer something about where a molecule is, the environment it's in, what's going on. So actually the key is being able to both covalently and chemically attach those dimolecules, but then also to be able to collect that light in an efficient way. So this actually is a field, so-called single molecule microscopy, which has developed over the past, say, 20 years of trying to understand, not just how light interacts with matter, but also how that can lead to kind of biological interpretation inside living cells, for instance.

Segment:3 Super resolution in action with Lisa-Maria Needham.
LISA-MARIA NEEDHAM: One of the first things that we do is clean our slides, our glass cover slides. And that's really important because we're operating at such high resolutions that you want your signal to noise ratio to be really high. So in order to minimize the amount of background that you get, you have to make sure that your slides are really clean. And so we do that with argon plasma cleaners. So we put our slides in for-- depending on the experiment-- it can be up to an hour.
LISA-MARIA NEEDHAM: And then in the meantime if we're doing a cell experiment, we'd label our cells. And that can take anything up to around 40 minutes. So you'd label your cell with a dye. And then you'd have to do several washing steps and washing processes. And then while that's happening, you kind of have a few things on the go. And so in that sort of time, any kind of waiting time, I'd come and turn the microscope on.
LISA-MARIA NEEDHAM: I'd check that all the lasers were functioning properly. And then I would clean the objective. That's something else that's really important. So we use most often oil immersion objectives. And so we have to clean them, because oil residues are typically quite dirty. So if you leave any residue on there for a while, you can, one, corrode the objective. And two, you can get kind of background and unwanted sort of aberrations from having kind of old oil, too much oil, things like that.
LISA-MARIA NEEDHAM: So we always make sure the objective's really clean. And then when it's ready I would put my cell sample on to the microscope. I would have a look with the white light, just to make sure that I could see the cells, make sure I was in the right kind of focal plane for whatever it is that I'm looking at. So, for example, we do a lot of experiments. Sometimes we want to look at the membrane of a cell.
LISA-MARIA NEEDHAM: So you'd want to make sure that you've kind of got a uniform illumination of the membrane. And often we do that with a TIRF illumination. And sometimes we want to look at the nucleus of a cell. And we'd use what's called oblique angle illumination. So rather than having the kind of evanescent wave of TIRF, which only illuminates the bottom sort of 150 nanometers of the sample, you'd bring up your beam so it cuts through.
LISA-MARIA NEEDHAM: So you don't get as good a signal to noise as you do with TIRF, but it's still pretty high. And so you bring it through, and you illuminate a section of the cell. And so sometimes we use that mode of illumination. So I just check that everything kind of looks to standard with that. And then I collect the data, depending on what I'm doing. So if you're doing a super resolution experiment, basically you need to get enough localizations over time without any of these flourophores overlapping with each other.
LISA-MARIA NEEDHAM: And that's really important. So basically you gain very high spatial resolution by sacrificing temporal resolution. So a super resolution experiment can take up to hours, depending on what you're doing. So I mean, it can take minutes, but it depends on the mode that you're looking at. So you typically take a few thousand frames, maybe, say, 30 milliseconds per frame.
LISA-MARIA NEEDHAM: So that can take a few minutes normally. And so you basically do that over, and over, and over again until you get all the data that you need. And then yeah. And then it's analysis and other kind of post processing things to get your image and whatever data it is you're extracting. And that's pretty much a typical super resolution experiment.
Segment:4 What direction is this field moving in?.
STEVEN LEE: The areas that it's going to be applied to I think are kind of huge. And so, for instance, in our lab we have a diverse set of interesting questions, things like, as I said, about neurodegeneration, about autoimmune disease. But also questions about cancer. All of these are accessible to these types of technologies. The question really is not-- I don't think there's any specific areas this is moving in.
STEVEN LEE: I think this is a really exciting time for super resolution, because it's really trying to find where can it be most useful? And there's been a few huge discoveries, whereby we're not just seeing things clearer. There've actually been scientific discoveries that have been made possible because of the technology.