Super-Resolution Imaging of AMPA Receptors Trafficking at Live Synapses

Synapses are the fundamental structures involved in signal transmission in neurons. Due to its sub-micron size, spatial resolution has been a major limiting factor in investigating the structure and dynamics of the synapses using light microscopy. We have developed a novel approach, based on light microscopy, combining three-dimensional single particle tracking with super-resolution imaging (PALM) to overcome this limitation. The technique has been successfully applied on live neurons to visualize the diffusion behavior of AMPA receptors near synapses, with sub-diffraction-limit resolution. We first used commercially available quantum dots with 20-30 nm in size to label the AMPA receptors as markers for single particle tracking and a photoactivable protein attached to post-synaptic protein Homer1 as a marker for PALM. As a result, we are able to simultaneously localize the post-synaptic density in three dimensions and observe the dynamic changes of AMPA receptors that move into, and out of, the synaptic area. We then changed our probe to a much smaller quantum dot, only 6 nm in diameter. With this smaller probe, different diffusion behavior of AMPA receptors is observed, which indicates the bigger quantum dots may have hindered the AMPA receptors' accessibility to the synaptic cleft.