Ratiometry of transmembrane voltage-sensitive fluorescent dye emission in hearts.

Transmembrane voltage-sensitive fluorescence measurements are limited by baseline drift that can obscure changes in resting membrane potential and by motion artifacts that can obscure repolarization. Voltage-dependent shift of emission wavelengths may allow reduction of drift and motion artifacts by emission ratiometry. We have tested this for action potentials and potassium-induced changes in resting membrane potential in rabbit hearts stained with di-4-ANEPPS [Pyridinium, 4-(2-(6-(dibutylamino)-2-naphthalenyl) ethenyl)-1-(3-sulfopropyl)-, hydroxide, inner salt] using laser excitation (488 nm) and a two-photomultiplier tube system or spectrofluorometer (resolution of 500-1,000 Hz and <1 mm). Green and red emissions produced upright and inverted action potentials, respectively. Ratios of green emission to red emission followed action potential contours and exhibited larger fractional changes than either emission alone (P < 0.001). The largest changes and signal-to-noise ratio (signal/noise) were obtained with numerator wavelengths of 525-550 nm and denominator wavelengths of 650-700 nm. Ratiometry lessened drift 56-66% (P < 0.015) and indicated decreases in resting membrane potential. Ratiometry lessened motion artifacts and increased magnitudes of deflections representing phase-zero depolarizations relative to total deflections by 123-188% in intact hearts (P < 0.02). Durations of action potentials at different pacing rates, temperatures, and potassium concentrations were independent of whether they were measured ratiometrically or with microelectrodes (P > or = 0.65). The ratiometric calibration slope was 0.017/100 mV and decreased with time. Thus emission ratiometry lessens the effects of motion and drift and indicates resting membrane potential changes and repolarization.

A telemetry system for the study of spontaneous cardiac arrhythmias.

The characteristics of spontaneous cardiac arrhythmias leading to sudden cardiac death are largely unknown. To study arrhythmias in animal models, an eight-channel implantable radio telemetry system has been developed to record continuously cardiac electrograms over a period of weeks to months, with maintenance restricted to changing batteries. The inputs are connected in a unipolar manner. Each channel has a gain of fifty and is AC coupled, band limited to 0.07-260 Hz. The signals are digitized with 12 bits resolution at 1000 samples/s. The amplifiers, analog-to-digital converter, and control logic are packaged in an implantable unit. An umbilical cable is passed through the skin to an external backpack unit for power and data transmission. A custom serial interface card, a PC/104 form factor 25-MHz 80386-based single-board computer with a PCMCIA wireless local area network (WLAN) card, and battery power supply make up the backpack. Data are read into the parallel port of the computer, buffered, then transmitted over the WLAN to the laboratory network where it can be analyzed and archived. Approximately 12 h of 14,000 bytes/s data can be collected with each set of batteries. The system is suitable for continuous monitoring of animal models of spontaneous arrhythmias and sudden cardiac death.

Transmembrane potential changes caused by monophasic and biphasic shocks.

Transmembrane potential change (DeltaVm) during shocks was recorded by a double-barrel microelectrode in 12 isolated guinea pig papillary muscles. After 10 S1 stimuli, square-wave S2 shocks of both polarities were given consisting of 10-ms monophasic and 10/10-ms and 5/5-ms biphasic waveforms that created potential gradients from 1.1 +/- 0.3 to 11.9 +/- 0.4 V/cm. S2 shocks were applied with 30, 60- to 70-, and 90- to 130-ms S1-S2 coupling intervals so that they occurred during the plateau, late portion of the plateau, and phase 3 of the action potential, respectively. Some shocks were given across as well as along the fiber orientation. The shocks caused hyperpolarization with one polarity and depolarization with the opposite polarity. The ratio of the magnitude of hyperpolarization to that of depolarization at the three S1-S2 coupling intervals was 1.5 +/- 0.3, 1.1 +/- 0.2, and 0.5 +/- 0.2, respectively. DeltaVm during the shock was significantly greater for the monophasic than for the two biphasic shocks. The prolongation of total repolarizing time (TRT) was significantly greater for monophasic (119.8 +/- 19.1%) and 10/10-ms biphasic (120.5 +/- 18.2%) than for 5/5-ms biphasic (113.0 +/- 12.9%) waveforms. The dispersion of the normalized TRT between instances of hyperpolarization and depolarization caused by the two shock polarities was 7.4 +/- 7.1% for monophasic, 3.0 +/- 4.1% for 10/10-ms biphasic, and 2.8 +/- 3.1% for 5/5-ms biphasic shocks (P < 0.05 for monophasic vs. biphasic). Shock fields along fibers produced a larger DeltaVm and prolongation of TRT than those across fibers. We conclude that 1) a change in shock polarity causes an asymmetrical change in membrane polarization depending on shock timing; 2) the 5/5-ms biphasic waveform causes the smallest DeltaVm, prolongs repolarization the least, and causes the smallest polarity-dependent dispersion; and 3) the changes in transmembrane potential and repolarization are influenced by fiber orientation.

Pion treatment procedures and verification techniques.

Procedures and techniques developed for the negative pi-meson (pion) radiotherapy program at the Los Alamos Meson Physics Facility, Los Alamos, NM, are reviewed and described. A particular pion patient is followed through the entire planning and treatment sequence to describe CT scanning procedures, bolus and collimator and treatment techniques developed to minimize positioning errors (less than 5 mm). Comparison of 2-D and 3-D isodose calculations developed at Los Alamos showed differences of less than 10% attributable to multiple scattering effects and the computational models used. Treatment verification methods using in vivo ion chamber dosimetry generally confirmed the prescribed dose delivery within 10% and using TLD within 18%.