Going beyond 2D: following membrane diffusion and topography in the IgE-Fc[epsilon]RI system using 3-dimensional tracking microscopy

Nathan P. Wells; Guillaume A. Lessard; Mary E. Phipps; Peter M. Goodwin; Diane S. Lidke; Bridget S. Wilson; James H. Werner

doi:10.1117/12.809412

24 February 2009 Going beyond 2D: following membrane diffusion and topography in the IgE-Fc[epsilon]RI system using 3-dimensional tracking microscopy

Nathan P. Wells, Guillaume A. Lessard, Mary E. Phipps, Peter M. Goodwin, Diane S. Lidke, Bridget S. Wilson, James H. Werner

Author Affiliations +

Proceedings Volume 7185, Single Molecule Spectroscopy and Imaging II; 71850Z (2009) https://doi.org/10.1117/12.809412
Event: SPIE BiOS, 2009, San Jose, California, United States

Abstract

The ability to follow and observe single molecules as they function in live cells represents a major milestone for molecular-cellular biology. Here we present a tracking microscope that is able to track quantum dots in three dimensions and simultaneously record time-resolved emission statistics from a single dot. This innovative microscopy approach is based on four spatial filters and closed loop feedback to constantly keep a single quantum dot in the focal spot. Using this microscope, we demonstrate the ability to follow quantum dot labeled IgE antibodies bound to FcεRI membrane receptors in live RBL-2H3 cells. The results are consistent with prior studies of two dimensional membrane diffusion (Andrews et al., Nat. Cell Biol., 10, 955, 2008). In addition, the microscope captures motion in the axial (Z) direction, which permits tracking of diffusing receptors relative to the "hills and valleys" of the dynamically changing membrane landscape. This approach is uniquely capable of following single molecule dynamics on live cells with three dimensional spatial resolution.

Citation Download Citation

Nathan P. Wells, Guillaume A. Lessard, Mary E. Phipps, Peter M. Goodwin, Diane S. Lidke, Bridget S. Wilson, and James H. Werner "Going beyond 2D: following membrane diffusion and topography in the IgE-Fc[epsilon]RI system using 3-dimensional tracking microscopy", Proc. SPIE 7185, Single Molecule Spectroscopy and Imaging II, 71850Z (24 February 2009); https://doi.org/10.1117/12.809412

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available