Super resolution microscopy

Super resolution microscopes can be used to characterize the spatial organization of the phosphorylating protein on the membrane of T-cells. The resolution of a microscope refers to the ability to distinguish two adjacent fluorophores, defined by the distance between the two closest fluorophores. Super resolution microscopy allows us to image beyond the diffraction limit. This is achieved by single-molecule microscopy, in which individual molecules are isolated at high density. Individual molecules are isolated by photoactivating them in series and subsequently bleach the photo-activatable fluorescent protein. Adjacent molecules will likely not be activated simultaneously with the photo-activatable fluorescent proteins, consequently a closer distance of adjacent molecules can be distinguished.

In this video you can see the individual phosphorylating protein-molecules being photo activated and subsequently bleached away. With these movies we can quantify the spatial organisation of the protein with and without the drugs.