Abstract
Voltage imaging is an important complement to traditional methods for probing cellular physiology, such as electrode-based patch clamp techniques. Unlike the related Ca(2+) imaging, voltage imaging provides a direct visualization of bioelectricity changes. We have been exploring the use of sulfonated silicon rhodamine dyes (Berkeley Red Sensor of Transmembrane potential, BeRST) for voltage imaging. In this study, we explore the effect of converting BeRST to diEt BeRST, by replacing the dimethyl aniline of BeRST with a diethyl aniline group. The new dye, diEt BeRST, has a voltage sensitivity of 40% ΔF/F per 100 mV, a 33% increase compared to the original BeRST dye, which has a sensitivity of 30% ΔF/F per 100 mV. In neurons, the cellular brightness of diEt BeRST is about 20% as bright as that of BeRST, which may be due to the lower solubility of diEt BeRST (300 μM) compared to that of BeRST (800 μM). Despite this lower cellular brightness, diEt BeRST is able to record spontaneous and evoked action potentials from multiple neurons simultaneously and in single trials. Far-red excitation and emission profiles enable diEt BeRST to be used alongside existing fluorescent indicators of cellular physiology, like Ca(2+)-sensitive Oregon Green BAPTA. In hippocampal neurons, simultaneous voltage and Ca(2+) imaging reveals neuronal spiking patterns and frequencies that cannot be resolved with traditional Ca(2+) imaging methods. This study represents a first step toward describing the structural features that define voltage sensitivity and brightness in silicon rhodamine-based BeRST indicators.