Single-Molecule imaging with Bruno da Rocha-Azevedo
Single-Molecule imaging with Bruno da Rocha-Azevedo
BRUNO DA ROCHA-AZEVEDO: Hi, my name is Bruno da Rocha-Azevedo. I am a senior research scientist at the Department of Biophysics at UT Southwestern Medical Center in Dallas, Texas. [MUSIC PLAYING] So I came here to present my data on single medical imaging of the receptor VEGFR-2, which is a very important receptor for angiogenesis. So what we do in our lab is to study the spatiotemporal dynamics of this receptor. So, with live cells, we're able to image single molecules and how they move and how they organize on the surface of those important cells for angiogenesis.
Segment:2 Single molecule imaging and analysis techniques.
BRUNO DA ROCHA-AZEVEDO: [MUSIC PLAYING] The way we do to make sure that we have as close to the in vivo situation possible, we have primary endothelial cells, which are not transformed or anything like that. We label the endogenous receptor VEGFR-2 using Fab fragments from a neutral VEGFR-2 antibody. And then we image those cells using TIRF at 37 degrees, which is the temperature that those cells live.
BRUNO DA ROCHA-AZEVEDO: And then, after that we get those movie streams, we go towards a computational pipeline. So we detect those single molecule particles using a Gaussian mixing model. And then we track the movements of those receptors, those particles, over time. And, after we get those tracks, we actually classify the tracks, and we classify the tracks in two types based on their diffusion coefficients.
BRUNO DA ROCHA-AZEVEDO: We classify them as mobile and immobile confined. And then, with this data, we can actually now respond a lot of questions about the role of the ligand in the organization of those receptors, in terms of diffusion or mobility, and also how they actually organize themselves, like clustering or dimerization and that sort of events.
Segment:3 Potential pitfalls in single molecule imaging.
BRUNO DA ROCHA-AZEVEDO: [MUSIC PLAYING] The hardest thing about single molecule imaging, in my opinion, is to have the right level of labeling because, if you have too much labeling, you're not able to detect single molecule particles.
BRUNO DA ROCHA-AZEVEDO: And then you lose the idea of what is a single molecule because you just have too much labeling. That's one problem. The other problem is also, if you label too few, and then you don't have particles enough to be detected, and you cannot actually see the interactions of the receptors because you have one label here, one label here, and then things just don't move. You cannot actually measure the interactions.
BRUNO DA ROCHA-AZEVEDO: So you have to really, each lab and each type of protein, you have to calibrate a proper level of labeling, which sometimes is between 15%, 20% of labeling of the protein that you're working with. So that I think is the hardest thing, to find a proper level of labeling to be able to get a proper assessment of single molecule level.
Segment:4 Impact of study of VEGFR-2 in Cell Biology.
BRUNO DA ROCHA-AZEVEDO: [MUSIC PLAYING] In terms of I think it is very impactful because most of the work that is being done with VEGFR-2 is mostly based on static situations like so you have fixed cells, or you have-- you make lysates for Western blot and biochemistry assays.
BRUNO DA ROCHA-AZEVEDO: So you don't have actually the sense of what's going on while the cell is alive. And, if we think about angiogenesis, the location and where is the receptor and how the receptor is moving I think is critical for cell making decision of making a new blood vessel. So, of course, this is looking at the big picture, but working with live cells just give you the proper assessment of what is going on with the cell at that moment and then, especially, with this specific receptor.
Segment:5 Single-molecule imaging in 3D tissues.
BRUNO DA ROCHA-AZEVEDO: [MUSIC PLAYING] So, because we use TIRF microscopy, which is a very powerful imaging system, but, however, it just gets a very small level of surface interactions, and most of our tissues are in 3D, single molecule imaging in 3D is still a little challenging because single molecule imaging, especially on the types of the ones we do, the fluorescence is very dim.
BRUNO DA ROCHA-AZEVEDO: So, when you have this in a 3D setting, it's very hard to have. So I think the next technological step will be when we have actually the capacity to do high level, high resolution 3D single molecule imaging of live cells. So that would be the ideal, but I think we're going to get there. It's just a matter of time.
Segment:6 Single-molecule imaging in 5 years' time.
BRUNO DA ROCHA-AZEVEDO: [MUSIC PLAYING] I really think that, actually, in the next five years, we are getting over the 2D, 3D transition. I think there are a lot of scientists improving 3D imaging like so many in the world doing that. And, also, the labeling part, so there are better fluorophores coming up that are much brighter. They're much more brighter.
BRUNO DA ROCHA-AZEVEDO: You can image cells for longer. So, if you can image cells for longer, you can have much longer videos of live cell imaging, maybe hours imaging the same cell. And then you can have a much better picture of the full behavior of a receptor or any other protein on a cell.
Segment:7 Dreams for single-molecule imaging.
BRUNO DA ROCHA-AZEVEDO: [MUSIC PLAYING] I would love to have a system that I can do, actually, single molecule imaging and tracking in a 3D setting, that I can actually make blood vessels in vitro and actually image the cells and how the receptors are behaving in, actually, very, very close to the in vivo setting.
BRUNO DA ROCHA-AZEVEDO: That would be ideal. Or, actually, even better, even better is actually having in vivo image in a single molecule level because then you can image on a mouse or a rabbit and actually see the receptors happening in real time. That would be fantastic. [MUSIC PLAYING]