Differential evanescence nanometry: Live-cell fluorescence measurements with 10-nm axial resolution on the plasma membrane

We present a method to resolve components within a diffraction-limited object by tracking simultaneously the average axial positions of two different sets of fluorescent molecules within it. The axial positions are then subtracted from each other to determine the separation of the two sets of fluorophores. This method follows the dynamic changes in the separation of the two sets of fluorophores with freely rotating dipoles using sequential acquisitions with total internal reflection and wide-field illumination, and it can be used to measure the formation of small structures on living cells. We have verified that we can achieve a resolution of 10 nm, and we have used the method to follow the location of clathrin and its adaptor AP-2 as they are recruited to a diffraction-limited coated pit during its assembly at the plasma membrane. We find a gradually increasing axial separation between the centroids of clathrin and AP-2 distribution, up to a final value of 30 nm just before coated-pit pinching and formation of the coated vesicle.