Action potential recordings from isolated guinea pig ventricular cells in the whole-cell recording mode were used to study the toxic and photodynamic properties of the voltage-sensitive fluorescent dye di-4-ANEPPS. Staining of the cardiomyocytes with di-4-ANEPPS (30 or 60 microM; 10 min) did not alter the action potential shape. When the stained cells were illuminated (1W/cm2) severe effects on the action potential were observed. There was a prolongation of the action potential duration, occurrence of early afterdepolarizations, reduction of the membrane resting potential and eventually inexcitability. Addition of the antioxidant catalase (100 IU/ml) to the extracellular solution delayed the onset of these effects, suggesting that reactive-oxygen-intermediates take part in di-4-ANEPPS induced photodynamic damage. Since di-4-ANEPPS is a very important tool for optical membrane potential recordings in heart tissue and single cardiomyocytes catalase might be useful in suppressing photodynamic damage during optical potential recordings.

An especially designed setup which consists of an inverted fluorescence microscope, an argon ion laser and a photodiode array system permits membrane potential monitoring in isolated guinea-pig ventricular cardiomyocytes, stained with the voltage-sensitive dye di-4-ANEPPS, which responds linearly with relative fluorescence changes (delta F/F) approximately -8% per 100 mV. About a dozen measuring spots covering a single cell were simultaneously monitored with a spatial and temporal resolution of 15 microns and about 20 microseconds, respectively. In general, the rising phases of the action potentials within a single cell were highly synchronized (i.e. all upstroke velocities peaked within about 20 microseconds); however, in one cell (out of 25 examined) significant (P < 0.05) time lags exceeding the signal-dependent time resolution were also found. Experiments, simultaneously performed with our optical system and a widely used patch-clamp setup, revealed a slowed and delayed response of the clamp amplifier depending on the cell access resistance. Optical monitoring during whole-cell voltage-clamping demonstrated the influence of graduated series resistance compensation. When field stimulation was used, our results clearly demonstrated the spatially dependent polarization of the cell membrane during the stimulus, as well as a highly synchronized upstroke development. Slight differences in the maximum upstroke velocities within a single cell were also found and were basically in agreement with mathematical models.

The rising phase of rat cardiac action potentials was measured in physiological solutions using the voltage-sensitive dye RH 237. A newly designed optical system and an argon ion laser for excitation allowed measurements without averaging over small areas (20-90 microns diameter) with high time resolution (response time 10-90%, 0.12 ms). The mean value of the fractional change in the fluorescence signal was approximately 3%/100 mV. The signal-to-noise ratio was approximately 60 rms (spot diameter 70 microns) allowing signal differentiation after digital filtering. Multiple measurements within the same spot showed a decrease in the fractional fluorescence change of 20 to 25% after 45 min without changes in the shape of the rising phase and with no measureable phototoxic effects. The optically measured rising phases showed rise times significantly (P less than 0.01) shorter and maximum upstroke velocities equal to or most often greater than those obtained with microelectrode techniques. Comparing simultaneous optical and electrical measurements within the same spot the microelectrode signal was often slightly delayed. This refined system seems well suited to detect fast cellular electrical activities with time and space resolutions comparable or even superior to those obtained using microelectrode techniques.

Several styryl dyes were tested as fast optical probes of membrane action potentials in mammalian heart muscle tissue. After staining, atrial specimens were superfused in physiological salt solution, and fluorescence was excited by an argon ion laser. Excitation spot size on the surface of the preparation was 60 microns in diameter. Dyes RH 160, RH 237, and RH 421 performed excellently as fast fluorescent probes of cardiac membrane potential. Fractional fluorescence changes, delta F/F, due to the action potential were in the range 2 to 6% at 514.5 nm excitation. Rise times of the action potential onset detected with each of the dyes were less than 0.5 ms, which is as fast or even faster than microelectrode measurements (atria of the rat). Thus membrane potential changes could be monitored with high resolution in both time and space. Emission spectra from heart muscle preparations stained with these dyes were shifted to shorter wavelengths by 70 nm and more as compared to spectra of the dyes in ethanol solution. The fluorescence spectrum of RH 160 at resting potential and the spectrum recorded during the plateau phases of the action potential were measured and showed no difference within the spectral resolution. As can be concluded from measurements of fluorescence changes at different excitation wavelengths, electrochromism cannot be the only mechanism causing the potential response.

Many vital processes depend on the generation, changes, and conduction of cellular transmembrane potentials. Optical monitoring systems are well suited to detect such cellular electrical activities in networks of excitable cells and also tissues simultaneously at multiple sites. Here, an exceptionally fast array system (16 x 16 photodiodes, up to 4,000,000 samples per second, 12-bit resolution) for imaging voltage-sensitive dye fluorescence, permitted real time measurements of excitation patterns at a microscopic size scale (256 pixels within an area of 1.8-8 mm2), in rat cardiac muscle in vitro. Results emphasize a recent hypothesis for cardiac impulse conduction, based on cardiac structural complexities, that is contradictory to all continuous cable theory models.