Improved sensitivity in a modified Berkeley Red Sensor of Transmembrane potential

Voltage imaging is an important complement to traditional methods for probing cellular physiology, like electrode-based patch clamp techniques. Unlike the related Ca2+ 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, or 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 VF 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 BeRST, which may be due to the lower solubility of diEt BeRST (300 μM) compared to 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 Ca2+-sensitive Oregon Green BAPTA (OGB). In hippocampal neurons, simultaneous voltage and Ca2+ imaging reveals neuronal spiking patterns and frequencies that cannot be resolved with traditional Ca2+ imaging methods. This study represents a first step towards describing the structural features that define voltage sensitivity and brightness in silicon rhodamine-based BeRST indicators.