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Two-photon excitation of voltage sensitive dyes (VSDs) can measure rapidly changing electrophysiological signals deep within intact cardiac tissue with improved three-dimensional resolution along with reduced photobleaching and photo-toxicity compared to conventional confocal microscopy. Recently, a category of VSDs has emerged which records membrane potentials by photo-induced electron transfer. FluoVolt is a novel VSD in this category which promises fast response and a 25% fractional change in fluorescence per 100 mV, making it an attractive optical probe for action potential (AP) recordings within intact cardiac tissue. The purpose of this study was to characterize the fluorescent properties of FluoVolt as well as its utility for deep tissue imaging. Discrete tissue layers throughout the left ventricular wall of isolated perfused murine hearts loaded with FluoVolt or di-4-ANEPPS were sequentially excited with two-photon microscopy. FluoVolt loaded hearts suffered significantly fewer episodes of atrio-ventricular block compared to di-4-ANEPPS loaded hearts, indicating comparatively low toxicity of FluoVolt in the intact heart. APs recorded with FluoVolt were characterized by a lower signal-to-noise ratio and a higher dynamic range compared to APs recorded with di-4-ANEPPS. Although both depolarization and repolarization parameters were similar in APs recorded with either dye, FluoVolt allowed deeper tissue excitation with improved three-dimensional resolution due to reduced out-of-focus fluorescence generation under two-photon excitation. Our results demonstrate several advantages of two-photon excitation of FluoVolt in functional studies in intact heart preparations, including reduced toxicity and improved fluorescent properties. [ABSTRACT FROM AUTHOR]

Abstract: Neurons in the visual cortex receive input from the lateral geniculate nucleus (feed-forward), higher order visual areas (feedback) and local neurons in the surroundings (lateral interactions). Here we first briefly review the approximate timing and proportion of these three types of influences on the membrane potentials in visual areas 17, 18 and 19. Then we present original results from an independent component analysis of multiunit spike trains in the same visual areas to resolve the contribution from these three sources. We stimulated the visual cortex of the ferret with a small transient contrast square stimulus and recorded the multiunit activity in areas 17, 18 and 19 with single or multiple electrodes. The spike trains had three reproducible components having their maxima at 40, 55 and 105ms after the start of the presentation of the stimulus. The time course of the third component was significantly correlated with the population membrane potential in the supragranular layers of areas 17, 18 and 19. The first spike train component was interpreted as a feed-forward response, the second spike train component as driving the laterally spreading depolarization and the third spike train component as the firing caused by the lateral spreading- and the feedback depolarization. [Copyright &y& Elsevier]

Local electric fields modulate a variety of cellular processes and are particularly relevant in the nervous system. Signal propagation along an axon occurs through small (around 100 mV) rapid changes in the potential across the cell membrane. Experimental techniques capable of measuring voltage changes in cells, tissue, and animal models have been instrumental in understanding cell signaling in the nervous system. Optical approaches relying on fluorescent voltage sensors have emerged as a minimally invasive – and less technically demanding – alternative to traditional, electrode-based techniques. Combined with state-of-the-art optical microscopy techniques, luminescent voltage sensors allow voltage sensing at relevant timescales (sub-millisecond) and diffraction-limited spatial resolutions. Voltage-sensitive dyes and proteins are already well-established fluorescent voltage sensors, while semiconductor nanostructures and nitrogen vacancy-containing diamonds are attracting increasing attention due to their superior photoluminescent properties. In this review, we describe the working mechanism of the different classes of fluorescent voltage sensors, discuss their advantages and limitations, and compare their performance based on sensitivity and temporal resolution. Further, we discuss the more recent advances in optical voltage sensing and identify the knowledge gaps that need to be addressed for this technique to realize its full potential as a valid replacement to electrode-based sensing. • The state of the art in photoluminescent voltage sensing is discussed. • We describe the working mechanism of different photoluminescent voltage sensors. • We compare the performance of molecular and nanoparticle-based voltage sensors. • We summarize the approaches for calibrating sensors and their limitations. • We discuss the current limitations of photoluminescent voltage sensors. [ABSTRACT FROM AUTHOR]