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Clinicians, biologists, physicists, engineers, and computer scientists are coming together to better understand heart disease, which is currently the leading cause of death globally. Optical mapping, a high-speed fluorescence imaging technique that visualizes and measures key cardiac parameters such as action potentials, cytosolic calcium transients, and fibrillation dynamics, is a core research tool that has arisen from such interdisciplinary collaborations. In an effort to broaden its use, especially among clinical scientists and students, we developed a complete and low-cost optical mapping system, including a constant-flow Langendorff perfusion system, which minimizes the economic threshold to widespread use of this powerful tool in cardiac electrophysiology research. The system described here provides high spatiotemporal resolution data about action potentials, intracellular calcium transients and fibrillation wave dynamics in isolated Langendorff-perfused hearts (pigs and rabbits), relevant for translational research. All system components and software elements are fully disclosed with the aim of increasing the use of this affordable and highly versatile tool among clinicians, basic scientists and students wishing to tackle their own research questions with their own customizable systems. [ABSTRACT FROM AUTHOR]

Background: In cell biology, increasing focus has been directed to fast events at subcellular space with the advent of fluorescent probes. As an example, voltage sensitive dyes (VSD) have been used to measure membrane potentials. Yet, even the most recently developed genetically encoded voltage sensors have demanded exhausting signal averaging through repeated experiments to quantify action potentials (AP). This analysis may be further hampered in subcellular signals defined by small regions of interest (ROI), where signal-to-noise ratio (SNR) may fall substantially. Signal processing techniques like blind source separation (BSS) are designed to separate a multichannel mixture of signals into uncorrelated or independent sources, whose potential to separate ROI signal from noise has been poorly explored. Our aims are to develop a method capable of retrieving subcellular events with minimal a priori information from noisy cell fluorescence images and to provide it as a computational tool to be readily employed by the scientific community. Results: In this paper, we have developed METROID (Morphological Extraction of Transmembrane potential from Regions Of Interest Device), a new computational tool to filter fluorescence signals from multiple ROIs, whose code and graphical interface are freely available. In this tool, we developed a new ROI definition procedure to automatically generate similar-area ROIs that follow cell shape. In addition, simulations and real data analysis were performed to recover AP and electroporation signals contaminated by noise by means of four types of BSS: Principal Component Analysis (PCA), Independent Component Analysis (ICA), and two versions with discrete wavelet transform (DWT). All these strategies allowed for signal extraction at low SNR (− 10 dB) without apparent signal distortion. Conclusions: We demonstrate the great capability of our method to filter subcellular signals from noisy fluorescence images in a single trial, avoiding repeated experiments. We provide this novel biomedical application with a graphical user interface at 10.6084/m9.figshare.11344046.v1, and its code and datasets are available in GitHub at . [ABSTRACT FROM AUTHOR]

Acid-sensing ion channel 1a (ASIC1a) is involved in several pathologies, including neurodegenerative and neuroinflammatory disorders, stroke, epilepsy, and inflammatory pain. ASIC1a has been the subject of intense drug discovery programs devoted to the development of new pharmacological tools for its modulation. However, these efforts to generate new compounds have faced the lack of an efficient screening procedure. In the past decades, improvements in screening technologies and fluorescent sensors for the study of ion channels have provided new opportunities in this field. Unfortunately, ASIC1a is mainly a Na+ permeable channel and undergoes desensitization after its activation, two features that make the use of the available screening procedures problematic. We propose here a novel screening approach for the study of ASIC1a activity in full automation. Our method is based on the stimulation of ASIC1a-expressing cells by protons and the use of electrochromic fluorescent voltage sensors as a readout of ion channel activation. This method will prove to be useful for drug screening programs aimed at ASIC1a modulation. [ABSTRACT FROM AUTHOR]

Fast responding voltage sensitive dyes, RH421 and di-4-ASPBS, were used to study the electrogenic properties of plant plasma membrane proton pumps on sealed plasma membrane vesicles extracted by two-phase partitioning from Beta vulgaris and Avena sativa cv Swan root material. Fluorescence spectroscopy in the presence of the dye RH421 (10.8 nM) was sufficiently sensitive to detect electrogenic activity of the extracted plant vesicles. The dye detection system could detect inhibition of electrogenic activity of vesicles by vanadate (75 μM) and stimulation by nigericin (0.5 μM). The newly developed dye di-4-ASPBS was less sensitive to detecting the electrogenic proton pump activity. This study represents an important innovation in plant biophysics as this class of fast responding voltage sensitive dyes have never to our knowledge been used to study electrogenic proton pump activity derived from plant membranes and represents a novel approach for carrying out such studies. [ABSTRACT FROM AUTHOR]

Objective: Unidirectional conduction block (UCB) and reentry may occur as a consequence of an abrupt tissue expansion and a related change in the electrical load. The aim of this study was to evaluate critical dimensions of the tissue necessary for establishing UCB in heart cell culture. Methods: Neonatal rat heart cell cultures with cell strands of variable width emerging into a large cell area were grown using a technique of patterned cell growth. Action potential upstrokes were measured using a voltage sensitive dye (RH-237) and a linear array of 10 photodiodes with a 15 μm resolution. A mathematical model was used to relate action potential wave shapes to underlying ionic currents. Results: UCB (block of a single impulse in anterograde direction — from a strand to a large area — and conduction in the retrograde direction) occurred in narrow cell strands with a width of 15(SD 4) μm (1–2 cells in width, n = 7) and there was no conduction block in strands with a width of 31(8) μm (n = 9, P < 0.001) or larger. The analysis of action potential waveshapes indicated that conduction block was either due to geometrical expansion alone (n = 5) or to additional local depression of conduction (n = 2). In wide strands, action potential upstrokes during anterograde conduction were characterised by multiple rising phases. Mathematical modelling showed that two rising phases were caused by electronic current flow, whereas local ionic current did not coincide with the rising portions of the upstrokes. Conclusions: (1) High resolution optical mapping shows multiphasic action potential upstrokes at the region of abrupt expansion. At the site of the maximum decrement in conduction, these peaks were largely determined by the electrotonus and not by the local ionic current. (2) Unidirectional conduction block occurred in strands with a width of 15(4) μm (1–2 cells). [ABSTRACT FROM PUBLISHER]

We monitored optical signals from cortex stained with a voltage sensitive dye to study activity evoked by intracortical electrical stimulation. The objectives were to study the spatial and temporal spread of activity from intrinsic connections near the stimulating electrode and to develop a new technique to study extrinsic projections from striate cortex to extrastriate target areas. Various measures were made of the time course of the optical signal (latency, rise time, decay time, temporal summation, facilitation versus depression, and presence or absence of a slow undershoot); in general, these measures were found to vary significantly across different response positions, different experiments, and even different runs within the same experiment. The spatial distribution of responses near the stimulating electrode in striate cortex was usually elliptical and was most often elongated along the anterior-posterior axis, with a typical size (full width at 75% max) of 1.3 mm (anterior-posterior axis) by 0.75 mm (medio-lateral axis). In some cases, complex spatio-temporal patterns were observed, in which the position of the maximum optical signal shifted with time or split into multiple peaks. In eight experiments, a response focus was found in extrastriate cortex at an expected location within the lateromedial area (LM). The response focus in LM was typically about half the size of that in striate cortex. In some experiments we observed additional focal responses in the anterolateral visual area (AL). The extrastriate responses showed a significant delay (3-10 ms) in onset and time to peak relative to the striate response. The validity of this technique for determining extrinsic projections was tested in two types of experiments. In the first, stimulation from two electrodes in striate cortex generated response foci consistent with the known topographic organization of area LM. In the second, the optically measured response focus was shown to correlate with the histologically reconstructed projection of a chemical tracer injected near the site of stimulation. We discuss the chain of neurophysiological events that occur during and after focal electrical stimulation and how they relate to the observed optical signal. We conclude that direct passive responses were a small component of our signal, that the component due to action potentials in directly stimulated neurons should have occurred in the first 1-2 ms post stimulus and is small compared to the peak signal, and that overall our signals were probably dominated by a combination of asynchronously occurring action potentials and excitatory and inhibitory synaptic potentials. These results, together with the prospect of being able to trace many connections in a single experiment, indicate that the combination of optical recording and focal electrical stimulation provides a valuable means for analyzing structural and physiological aspects of cortical circuitry. [ABSTRACT FROM AUTHOR]

An optical method was used to measure action potentials from frog ventricle, in vitro, under normal physiological conditions with 0.5-1 mM Ca Ringer's solution. The approach presented in this paper involves a portable fluorimeter coupled to a multimode optical fiber running into a glass pipette ('optrode') to carry both excitation light to and fluorescence from the ventricle stained with the voltage sensitive dye di-4-ANEPPS. A suction technique was used to stabilize the optrode-tissue interface, significantly reducing motion artifacts from the beating ventricle. The typical fractional change in fluorescence intensity for an action potential was −9%. The optical recordings faithfully reproduced membrane action potentials as measured with microelectrode recordings. To confirm further the validity of our method we studied the effect of an increasing stimulation rate on the optical action potential. The amplitude of the action potential did not increase, and the change in action potential duration was similar to published results obtained with microelectrode recordings, suggesting that our optical action potentials are relatively free of motion artifacts. Finally, our optical recordings suggest that during anodal and cathodal point stimulation, the time course of membrane potential differs from that predicted simply by a passive cable model. [ABSTRACT FROM AUTHOR]