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

¹ Harvard Medical School, Cell Biology Department/CBRI

^* To whom correspondence should be addressed. E-mail: kirchhausen{at}crystal.harvard.edu.

Submitted on July 12, 2007
Revised on August 6, 2007
Accepted on 24 October 2007

	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.

Key Words: AP2, Axial Resolution, Clathrin, Endocytosis, Evanescence Nanometry, High Resolution Imaging