 So in our lab we're very interested in the molecular biology of neurons. For example in our model system we grow neurons associated from the brain of a rat on a dish. And the neurons grow on a dish and are very interesting. For example where certain proteins are, for example where interesting protein A. And because where the protein is very often implies the function of this protein. For example this protein A is in this places of the neuron. And the conventional immunofluorescent microscopy Eury takes an antibody labeling strategy where we have a primary antibody that targets a protein A and then we have a secondary antibody that targets the primary antibody. The secondary is fluorescent it's attached to a fluorophore. So in this case so the primary antibody it finds a protein A and the secondary antibody finds a primary antibody and is attached to a fluorophore. And then through this fluorophore we can visualize this protein A. Now this is all very well and good but then the problem is when you have two copies of protein A that are very close to each other and then you would get labels that are very close to each other. In that case you have two fluorophores that are very close to each other they would actually the fluorophores would actually fluoresce like this when they're next to each other. And that gives us an image under the microscope that looks like one big cloud of fluorescence when in fact that could be one, two or any number of copies of fluorophores within this cloud of fluorescence. Now this is the problem that we face with conventional microscopy method is called the diffraction limit. And a new technique that allows us to super resolve these crowded molecules it takes a different approach to this. What it does is it takes a video of these fixed neurons where in one frame so example in frame one we see one molecule fluoresce and the other molecule despite being there it does not fluoresce and in frame two you know in another frame for example frame two the other molecule fluoresce and the molecule right next to it it does not fluoresce. So what and then in these frames you can very quickly pinpoint very precisely pinpoint the position of each molecule that fluoresce and we combine these frames in the end you create an image reconstruct the image where you have all the molecules that you've labeled and these molecules are super resolved from each other. And so this at the moment we can routinely achieve a resolution of about 10 nanometers and the diffraction limit that we have discussed here is about 200 nanometers at the best and the reason why we want to go beyond 200 nanometer resolution is when you look at these neurons they have these type of structures called synapses. These are the biological structures that that's responsible for information transfer and storage in these neurons. Now these biological this is a synapse for example the synaptic cleft is only about 20 nanometer that's way below the diffraction limit and then the size of the diameter of a synapse can be only a few hundred nanometers. So that's very close to the diffraction limit as well so that's the reason why we need a technique that allows us to resolve molecules away from each other. Now coming back to the methodology of how do we achieve this kind of blinking so that you know in one frame this molecule fluoresce and another frame another molecule fluoresce. So one way to do this is instead of putting a fluorophore onto an antibody our antibody secondary antibody is still here is instead attached to a DNA oligo. Now what this DNA oligo allows is it will capture the complementary oligo that is fluorescent that carries the fluorophore. Now this complementary oligo when it is bound to the the the oligo that's on the antibody then the fluorophore is on the protein as well then you would see this molecule but because this DNA oligo is so short it will also dissociate after a while. The hybridization lasts about a second and once it dissociates so it went away again and this protein this copy of protein you can't see it anymore because it doesn't have fluorescence so it's this kind of on-off dynamics that creates this kind of blinking effect that causes molecule to be flow to fluoresce in one frame but not anymore in another frame and this technique that that creates this kind of blinking and that eventually allows us to resolve molecules at this high resolution this copy may paint