Development of Fluorescent and Bioluminescent Hybrid Voltage Imaging Strategies for Voltage Sensitive Dyes
- Benlian, Brittany
- Advisor(s): Miller, Evan W
Abstract
Understanding the broader implications and functions of membrane potential in the brain,heart, and body requires accurate and simultaneous membrane-associate voltage measurements on a large array of single cells. Achieving high spatial resolution and millisecond time resolution with minimal perturbation to live cells is a long-standing challenge, especially within the field of neurobiology. Direct imaging of membrane potential changes with fluorescence-based voltage indicators has become a powerful and most popular approach to mapping electrical changes yet several challenges addressing indicator speed, brightness, sensitivity, and localization remain. The Miller lab focuses on addressing these issues using a relatively new class of small molecule voltage indicators, VoltageFlours, that depend on a photo-induced electron transfer (PeT) mechanism. The intramolecular electron transfer modulates fluorescence intensity based on the surrounding electric field and results in sub-microsecond response kinetics with high voltage sensitivity. As with all organic indicators, the cellular spatial resolution in dense tissue samples is limited since VoltageFluors indiscriminately stain all membrane types. Our group has worked on addressing this limitation by combining VoltageFluors with genetically encoded self-labeling enzymes like the well-known Halo- and SNAP-tag proteins. Portions of this dissertation will focus specifically on the SNAP-tag covalent tethering approach with fluorescein and rhodamine-based VoltageFluors. The remainder of this thesis is primarily focused on a new branch of voltage imaging using bioluminescence which has never been explored within our group. Fluorescent indicators are dependent on externally applied illumination leading to unavoidable light-induced damage, especially at shorter wavelengths, which alters cell physiology and confounds data. There are relatively few examples of bioluminescent indicators relative to fluorescent indicators, and even fewer that are voltage sensitive. This dissertation will explore the development of the first ever quenching bioluminescent voltage indicator, limitations to single cell bioluminescent voltage imaging, and other potential approaches to functionalize bioluminescence at the membrane.