Differential Evanescence Nanometry: Live Cell Fluorescence Measurements with 10 nm Axial Resolution on the Plasma Membrane

@article{citeulike:1925021, abstract = {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 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 prior to coated pit pinching and formation of the coated vesicle. 10.1529/biophysj.107.117234}, author = {Saffarian, Saveez and Kirchhausen, Tomas }, citeulike-article-id = {1925021}, doi = {10.1529/biophysj.107.117234}, journal = {Biophys. J.}, keywords = {imaging, superresolutiontirf}, month = {November}, pages = {biophysj.107.117234+}, posted-at = {2007-11-28 21:55:13}, priority = {2}, title = {Differential Evanescence Nanometry: Live Cell Fluorescence Measurements with 10 nm Axial Resolution on the Plasma Membrane}, url = {http://dx.doi.org/10.1529/biophysj.107.117234}, year = {2007} }

TY - JOUR ID - citeulike:1925021 L3 - citeulike-article-id:1925021 TI - Differential Evanescence Nanometry: Live Cell Fluorescence Measurements with 10 nm Axial Resolution on the Plasma Membrane JF - Biophys. J. SP - biophysj.107.117234 N2 - 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 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 prior to coated pit pinching and formation of the coated vesicle. 10.1529/biophysj.107.117234 KW - imaging KW - superresolutiontirf AU - Saffarian, Saveez AU - Kirchhausen, Tomas PY - 2007/11/09/ UR - http://dx.doi.org/10.1529/biophysj.107.117234 ER -