Light-mediated silencing and stimulation of cardiac excitability, an important complement to electrical stimulation, promises important discoveries and therapies. To date, cardiac optogenetics has been studied with patch-clamp, multielectrode arrays, video microscopy, and an all-optical system measuring calcium transients. The future lies in achieving simultaneous optical acquisition of excitability signals and optogenetic control, both with high spatio-temporal resolution. Here, we make progress by combining optical mapping of action potentials with concurrent activation of channelrhodopsin-2 (ChR2) or halorhodopsin (eNpHR3.0), via an all-optical system applied to monolayers of neonatal rat ventricular myocytes (NRVM). Additionally, we explore the capability of ChR2 and eNpHR3.0 to shape action-potential waveforms, potentially aiding the study of short/long QT syndromes that result from abnormal changes in action potential duration (APD). These results show the promise of an all-optical system to acquire action potentials with precise temporal optogenetics control, achieving a long-sought flexibility beyond the means of conventional electrical stimulation.

The Kryptopterus bicirrhis (glass catfish) is known to respond to electromagnetic fields (EMF). Here we tested its avoidance behavior in response to static and alternating magnetic fields stimulation. Using expression cloning we identified an electromagnetic perceptive gene (EPG) from the K. bicirrhis encoding a protein that responds to EMF. This EPG gene was cloned and expressed in mammalian cells, neuronal cultures and in rat’s brain. Immunohistochemistry showed that the expression of EPG is confined to the mammalian cell membrane. Calcium imaging in mammalian cells and cultured neurons expressing EPG demonstrated that remote activation by EMF significantly increases intracellular calcium concentrations, indicative of cellular excitability. Moreover, wireless magnetic activation of EPG in rat motor cortex induced motor evoked responses of the contralateral forelimb in vivo. Here we report on the development of a new technology for remote, non-invasive modulation of cell function.

SummaryRatiometric quantification of CFP/YFP FRET enables live-cell time-series detection of molecular interactions, without the need for acceptor photobleaching or specialized equipment for determining fluorescence lifetime. Although popular in widefield applications, its implementation on a confocal microscope, which would enable sub-cellular resolution, has met with limited success. Here, we characterize sources of optical variability (unique to the confocal context) that diminish the accuracy and reproducibility of ratiometric FRET determination and devise practical remedies. Remarkably, we find that the most popular configuration, which pairs an oil objective with a small pinhole aperture, results in intractable variability that could not be adequately corrected through any calibration procedure. By quantitatively comparing several imaging configurations and calibration procedures, we find that significant improvements can be achieved by combining a water objective and increased pinhole aperture with a uniform-dye calibration procedure. The combination of these methods permitted remarkably consistent quantification of sub-cellular FRET in live cells. Notably, this methodology can be readily implemented on a standard confocal instrument, and the dye calibration procedure yields a time savings over traditional live-cell calibration methods. In all, identification of key technical challenges and practical compensating solutions promise robust sub-cellular ratiometric FRET imaging under confocal microscopy.