RESUMEN
All optical neurophysiology allows manipulation and readout of neural network activity with single-cell spatial resolution and millisecond temporal resolution. Neurons can be made to express proteins that actuate transmembrane currents upon light absorption, enabling optical control of membrane potential and action potential signalling. In addition, neurons can be genetically or synthetically labelled with fluorescent reporters of changes in intracellular calcium concentration or membrane potential. Thus, to optically manipulate and readout neural activity in parallel, two spectra are involved: the action spectrum of the actuator, and the absorption spectrum of the fluorescent reporter. Due to overlap in these spectra, previous all-optical neurophysiology paradigms have been hindered by spurious activation of neuronal activity caused by the readout light. Here, we pair the blue-green absorbing optogenetic actuator, Chronos, with a deep red-emitting fluorescent calcium reporter CaSiR-1. We show that cultured Chinese hamster ovary cells transfected with Chronos do not exhibit transmembrane currents when illuminated with wavelengths and intensities suitable for exciting one-photon CaSiR-1 fluorescence. We then demonstrate crosstalk-free, high signal-to-noise ratio CaSiR-1 red fluorescence imaging at 100 frames s-1 of Chronos-mediated calcium transients evoked in neurons with blue light pulses at rates up to 20 Hz. These results indicate that the spectral separation between red light excited fluorophores, excited efficiently at or above 640 nm, with blue-green absorbing opsins such as Chronos, is sufficient to avoid spurious opsin actuation by the imaging wavelengths and therefore enable crosstalk-free all-optical neuronal manipulation and readout.
RESUMEN
Radiofrequency catheter ablation procedures are a first-line method of clinical treatment for atrial fibrillation. However, they suffer from suboptimal success rates and are also prone to potentially serious adverse effects. These limitations can be at least partially attributed to the inter- and intra- patient variations in atrial wall thickness, and could be mitigated by patient-specific approaches to the procedure. In this study, a modelling approach to optimising ablation procedures in subject-specific 3D atrial geometries was applied. The approach enabled the evaluation of optimal ablation times to create lesions for a given wall thickness measured from MRI. A nonliner relationship was revealed between the thickness and catheter contact time required for fully transmural lesions. Hence, our approach based on MRI reconstruction of the atrial wall combined with subject-specific modelling of ablation can provide useful information for improving clinical procedures.