Design, synthesis and application of long wavelength fluorophores for bioimaging

项目总结

Project Summary Neurons and cardiomyocytes utilize rapid changes in membrane potential for cellular signaling. Understanding how changes in membrane potential affect cell behavior across a population of neurons or cardiomyocytes is an important challenge in biology. Widely used techniques to measure membrane potential, either directly or indirectly, consist of patch clamp electrophysiology and calcium ion imaging agents. Using patch clamp electrophysiology to measure membrane potential results in accurate measurements of membrane potential on the timescale of the action potentials. However, these experiments are limited to a single cell membrane, which precludes using patch clamp electrophysiology to study larger cell populations. Calcium ion imaging agents are widely used, as it has a higher throughput and is less invasive than patch clamp electrophysiology. By measuring temporary changes in calcium ion concentrations, we are able to indirectly measure changes in membrane potential. Unfortunately, a major drawback in this technique is the long durations of calcium ion uptake (10-100 ms) found during a neuronal action potential, which is significantly longer than the actual action potential itself (1-2 ms). This our ability to understand cell behavior and how it relates to these rapid signaling events. To address this challenge, our group has developed several different fluorescent probes called VoltageFluor dyes to measure these rapid changes in membrane potential. The VoltageFluor dyes consist of a fluorescent xanthene core tethered to a hydrophobic molecular wire. The molecular wire localizes in the cell membrane while the xanthene core orients itself in the extracellular space. Using this system, we can measure rapid changes in membrane potential in large populations of cells on the same time scale as the action potentials themselves. While our previous VoltageFluors work as intended, we wish to develop new VoltageFluors that emit light in the near infrared. A red-shifted emission spectrum would allow these VoltageFluor dyes to be used in vivo as well as in conjunction with other indicators that emit light at <600 nm. To accomplish this goal, we will develop new VoltageFluor dyes that contain electron withdrawing groups in the xanthene backbone. In accordance with Dewar’s rule, the inclusion of electron withdrawing groups will result in a red-shifted absorption and emission spectra. This hypothesis is supported by computational studies we have undertaken. The planned syntheses of these xanthene cores will also be more robust and tolerant of various functional groups than the syntheses of our previous dimethylcarbon and dimethylsilicon VoltageFluor dyes. In comparison to our previously synthesized VoltageFluor dyes that utilize a dimethylcarbon and dimethylsilicon backbone in the xanthene core, the electron withdrawing groups found in the proposed VoltageFluors offer new functional handles that can easily be diversified. This expands the range of applications that these dyes can be utilized for, including tethering triplet state quenchers to the xanthene core and using genetic targeting techniques to localize these VoltageFluors in specific cells. We also anticipate that the facile diversification of the xanthene core will mean that these dyes will be more broadly applicable. By synthesizing these dyes without the molecular wire, we also anticipate that we will be able to use this technology in calcium ion imaging studies.