Person:

Hou, Jennifer

Single-molecule spectroscopy provides a wealth of information on the dynamics and interactions of complex biological molecules. Yet these measurements are extremely challenging, partly because Brownian motion prevents molecules in free solution from remaining stationary. Here we describe several techniques that our lab has developed for confining single molecules for optical spectroscopy. These alternatives to surface immobilization provide confinement that is gentle enough to minimize perturbations to the molecule, but strong enough to allow long-time imaging of single fluorescent molecules, often in the presence of a high fluorescent background.

I present applications of imaging and spectroscopy to understand mechanical, chemical, and electrical dynamics in biological materials. The first part describes the development and characterization of a protein-based fluorescent calcium and voltage indicator (CaViar). The far-red fluorescence of CaViar faithfully tracks the cardiac action potential in cardiomyocytes. CaViar's green fluorescence reports the resulting calcium transients. I demonstrated the applicability of CaViar in vivo with transgenic zebrafish designed to express CaViar in their hearts. Spinning disk confocal imaging allowed detailed three-dimensional mapping of simultaneous voltage and calcium dynamics throughout the heart of zebrafish embryos, in vivo, as a function of developmental stage. I tested the effect of channel blockers on voltage and calcium dynamics and discovered a chamber-specific transition from a calcium-dependent to a sodium-dependent action potential. I also describe a new measurement technique using a fluorescent voltage indicator to report absolute voltage via the indicator's temporal response.

Plasma membrane voltage is a fundamentally important property of a living cell; its value is tightly coupled to membrane transport, the dynamics of transmembrane proteins, and to intercellular communication. Accurate measurement of the membrane voltage could elucidate subtle changes in cellular physiology, but existing genetically encoded fluorescent voltage reporters are better at reporting relative changes than absolute numbers. We developed an Archaerhodopsin-based fluorescent voltage sensor whose time-domain response to a stepwise change in illumination encodes the absolute membrane voltage. We validated this sensor in human embryonic kidney cells. Measurements were robust to variation in imaging parameters and in gene expression levels, and reported voltage with an absolute accuracy of 10 mV. With further improvements in membrane trafficking and signal amplitude, time-domain encoding of absolute voltage could be applied to investigate many important and previously intractable bioelectric phenomena.