Establishment of Photo-Activated Localization Microscopy (PALM) for Imaging Signaling Complexes on the Surfaces of Cells

Photoactivated Localization Microscopy (PALM) permits visualization of single molecules inside cells at ~10 times the resolution of existing optical microscopes. The 10-50 nanometer resolution afforded by PALM is ideally suited to studying the organization of receptor signal transduction occurring on the surfaces of cells. Initial efforts presented here focused on cells expressing Fc receptor fused to photoactivatable green fluorescent protein (paGFP). A small fraction of the paGFP molecules were photoactivated using a 405 nm laser pulse, resulting in single molecule spots that could then be imaged using 488 nm laser excitation until they photobleached. Both lasers illuminated the sample through the objective lens using Total Internal Reflection (TIR) excitation which allowed for selective imaging of molecules within about 200 nm of the cell/coverglass interface. Iterations of alternating laser excitations results in thousands of images of single molecules. These images were analyzed using the ImageJ plugin, QuickPALM, which performs a least squares fit of a two dimensional Gaussian to each diffraction-limited (~250 nm wide) fluorescent spot captured in the PALM images (Henriques). This process recovers the most probable position to within ~40 nm accuracy of each paGFP molecule. This information is used to create the super resolution image of the cell. Ongoing efforts are aimed at establishing two-color PALM to determine the distribution of signaling receptors relative to key sub-resolution regulators of signaling. A better understanding of the nanoscale molecular activities within cells will allow for more in depth studies into many different fields including biophysics, molecular biology, and medicine