Research

Multicolor and time-resolved electron microscopy of cell signaling


We are developing new biophysical methods to investigate the nanoscale cellular organization of G protein-coupled receptor (GPCR) and neural signaling. We are currently working on these two technical directions:


1. Multicolor electron microscopy:

From the historical perspective, the state-of-the-art in multicolor electron microscopy is comparable to that of fluorescence microscopy 30 years ago, when lasers and detectors already existed, but the multicolor palette of fluorescent proteins, biocompatible dye conjugates, and quantum dots did not. We are developing a method for single-molecule multicolor electron microscopy that uses inorganic nanoparticles as luminescent protein tags. Under excitation by an electron beam, these nanoparticles emit light via a process known as cathodoluminescence. These “cathodophores” will permit observing both the cellular ultrastructure and individual proteins with nanoscale resolution in a single experiment.


2. Time-resolved cryo-vitrification:

Cell signaling involves orchestrated nanoscale motions of proteins and membranes triggered by a stimulus (e.g. addition of a drug). However, nanoscale imaging techniques lack the temporal resolution necessary to observe these motions. To recover this temporal information, we will develop methods that will allow cryo-vitrifying biological samples at ultrafast time delays following stimulation for subsequent super-resolved optical and electron imaging. These methods will (1) feature ultrafast stimulation-to-freezing timescales for mapping out some of the fastest cellular signaling events and (2) allow performing live-cell imaging immediately prior to vitrification for correlative light and electron microscopy experiments.

 

We will use the techniques we are developing to study the dynamic nanoscale organization of signaling biomolecules in purified samples, differentiated cells, and complex tissues.