Near Infrared Emitting Dye Di-4-ANBDQBS for Recording Action Potentials in Isolated Cardiomyocytes

Abstract

The use of voltage-sensitive dyes (VSDs) for noninvasive measurement of action potentials (AP) in isolated cells is limited by photodynamic damage. Here we tested a new red-shifted VSD Di-4-ANBDQBS as an optical AP (OAP) reporter. Methods: Guinea-pig ventricular myocytes were loaded with Di-4-ANBDQBS (18.4-73.6 μM), paced (CL=1-2s) and imaged (excitation, 660 nm laser, emission, >750 nm) in an inverted microscope (40x objective) using an EMCCD camera at 860 frames/s. The VSD signal was integrated over the cell image. Results: Limited exposure to laser (700 ms every 5 min) yielded OAPs with fast upstrokes (2-3 ms), stable duration (APD) and signal-to-noise (SNR) exceeding 20 for at least 30 min. The OAP faithfully followed the electrical AP obtained via patch pipette (Figure) yielding a high correlation between the respective APD measurements (R2=0.985). Longer laser exposures (10 s on/50 s off for 5 minutes) caused APD prolongation which could be alleviated by reducing laser power and/or dye concentration at the expense of reduced SNR. Conclusion: With careful consideration of laser exposure, Di-4-ANBDQBS shows significant promise for noninvasive AP recordings in cardiomyocytes with an acceptable SNR.View Large Image | View Hi-Res Image | Download PowerPoint Slide