Abstract
Background & aimsTwo-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. MethodsDiscrete 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. ResultsFluoVolt 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. ConclusionOur results demonstrate several advantages of two-photon excitation of FluoVolt in functional studies in intact heart preparations, including reduced toxicity and improved fluorescent properties.
Highlights
The heart is an electrically excitable organ, capable of transducing the rapid and coordinated spread of electrical excitability into cycles of mechanical contraction and relaxation, allowing efficient ejection of blood from the heart and subsequent filling of the chambers in preparation for the beat
Two-photon excitation images of a FluoVolt stained isolated cardiomyocyte and left ventricular epicardium stained with FluoVolt and Rhod-2AM are shown in Fig. 1A (i) and (ii), respectively
In the current study, using a previously published experimental approach combined with our whole heart experimental results, we have shown that FluoVolt allows electrophysiological recordings from deeper tissue layers than di-4-ANEPPS, most likely because less out-of-focus fluorescence is generated for a given degree of sample scattering with FluoVolt
Summary
The heart is an electrically excitable organ, capable of transducing the rapid and coordinated spread of electrical excitability into cycles of mechanical contraction and relaxation, allowing efficient ejection of blood from the heart and subsequent filling of the chambers in preparation for the beat. One category of VSDs that fulfills this requirement is the electrochromic dyes, whose voltage-sensing mechanism is based on a shift in spectral characteristics in response to changes in membrane electric field (Loew, 1996) This property allows recordings of APs with high signal-tonoise ratio (S/N) by ratioing the right and left sides of the emission curve, minimizing the distorting effects of motion artifacts, bleaching and uneven distribution of the dye (Loew, 1996). 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. Conclusion: 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
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