Synthesis and Development of Fluorescent Dyes for Imaging Neuronal Activity
- Ortiz, Gloria
- Advisor(s): Miller, Evan W
Fluorescent probes are noninvasive tools that can be used to map the electrical activity and synaptic communication of large networks of neurons. Fluorescent probes that bind Ca2+ or measure voltage changes translate activity into fluorescence signals with high spatial and temporal resolution. Current probes used for measuring neuronal activity utilize relatively short wavelengths of light. This dissertation discusses new synthetic methods to access sulfonated voltage sensitive dyes (VSDs) that are red-shifted and exhibit improved performance in cells, including greater photostability, voltage sensitivity, brightness, and signal to noise ratios. As most small molecule VSDs nonspecifically label cells, limiting their use in more complex tissues, chemical-genetic targeting approaches to target VSDs dyes for increased contrast and reduced background fluorescence will be discussed. We synthesize sulfonated carbofluorescein VoltageFluors (carboVFs) which utilize photoinduced electron transfer as a trigger to sense voltage in cells. CarboVFs display red-shifted profiles of >560 nm and high voltage sensitivities (up to 31% ΔF/F per 100 mV). The best performing carboVF, carboVF2.1(OMe).Cl, can be incorporated into an enzymatic, fluorogenic targeting strategy to specifically label cells. By replacing the oxygens in carboVFs with dimethylaminos, we accessed far-red TMCRh voltage indicators which possess excitation and emission profiles >620 nm, display improved photostability and reduced phototoxicity, and still maintain good voltage sensitivities (up to 18% ΔF/F per 100 mV). In particular, TMCRh.OMe rivals or outperforms reported far-red silicon rhodamine VSD, BeRST 1, when comparing signal to noise and ΔF/F in neurons, and has the potential to be used as a bright reporter of absolute membrane potential in non-excitable cells using fluorescence lifetime imaging. In addition to derivatizing the carbofluorescein scaffold to obtain new VSDs, this dissertation discusses a new synthetic route to access sulfonated silicon rhodamines and progress towards applying this strategy to carboxylated silicon rhodamines. Silicon rhodamines which bear a carboxyl instead of the typical sulfonate membrane anchor are important for the genetic-targeting of rhodamine-based VSD dyes to specific cells. We report the development of a genetically-targeted silicon rhodamine voltage indicator, isoBeRST-Halo, through a covalent labeling strategy using the HaloTag system. IsoBeRST-Halo maintains the high voltage sensitivity of the untargeted isoBeRSTs, while achieving high selectivity in cultured cells and cortical brain slice. To address the limitations of fast voltage imaging speeds and allow for the comprehensive mapping of neuronal networks, we sought to develop Ca2+ integrators based on the fluorogenic spirobenzopyran scaffold, calcium ligands, and a photoactivatable group. Various modifications to the spirobenzopyran scaffold will be discussed, as well as the synthesis and initial characterization of new calcium ligands. Together, this work demonstrates the power of synthetic chemistry to achieve highly selective, bright, and functional reporters of neuronal activity.