Multicolor and time-resolved electron microscopy of cellular architecture

We are developing new biophysical methods to investigate the nanoscale cellular organization with relevance to G protein-coupled receptor (GPCR) signaling, neural transmission, and microbial infections. We are currently pursuing two directions of biophysical method development to help us study these processes:

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. Cathodoluminescence microscopy is used routinely in semiconductor industry for characterization and analysis of solid-state devices. However, this light-matter interaction has been severely underexplored and underutilized with regard to biological applications. The reason for this gap is a lack of biocompatible "cathodophores" suitable for cathodoluminescence-based multicolor electron microscopy. Such 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. optogenetic excitation or addition of a drug). However, nanoscale imaging techniques lack the temporal resolution necessary to observe these motions. To recover this temporal information, we are developing cryo-plunging and high-pressure freezing methods that will allow vitrifying biological samples at ultrafast time delays (from sub-millisecond for cryo-plunging to a few milliseconds for high-pressure freezing) following stimulation for subsequent super-resolved optical and electron imaging. These methods will not only feature ultrafast stimulation-to-freezing timescales for mapping out some of the earliest events in cellular signaling, but will also allow performing live-cell imaging immediately prior to vitrification for correlative light and electron microscopy of active and inactive cellular states. The time-resolved cryo-vitrification techniques that we are developing effectively act as a “pause button” as they freeze and capture biological processes in space and time with ultrafast temporal resolution following a precisely delivered trigger pulse.


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