Pulsed local-field fluorescence microscopy: a new approach for measuring cellular signals in the beating heart (original) (raw)
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Frontiers in physiology, 2014
Dyssynchronous local Ca release within individual cardiac myocytes has been linked to cellular contractile dysfunction. Differences in Ca kinetics in adjacent cells may also provide a substrate for inefficient contraction and arrhythmias. In a new approach we quantify variation in local Ca transients between adjacent myocytes in the whole heart. Langendorff-perfused mouse hearts were loaded with Fluo-8 AM to detect Ca and Di-4-ANEPPS to visualize cell membranes. A spinning disc confocal microscope with a fast camera allowed us to record Ca signals within an area of 465 μm by 315 μm with an acquisition speed of 55 fps. Images from multiple transients recorded at steady state were registered to their time point in the cardiac cycle to restore averaged local Ca transients with a higher temporal resolution. Local Ca transients within and between adjacent myocytes were compared with regard to amplitude, time to peak and decay at steady state stimulation (250 ms cycle length). Image regis...
Journal of Pharmacological Sciences, 2003
Rapid-scanning cofocal microscopy has been applied to the analysis of early phase Ca 2+ transients in ventricular and atrial cardiomyocytes. On electrical stimulation of ventricular myocytes, Ca 2+ concentration begins to rise earliest at the Z-line level and becames uniform throughout the cytoplasm within about 10 ms after the onset of the action potential; transsarcolemmal Ca 2+ influx triggers Ca 2+ release from release sites on the junctional sarcoplasmic reticulum (SR) coupled to T-tubules at the Z-line throughout the cytoplasm. In atrial myocytes lacking the T-tubular network, transsarcolemmal Ca 2+ influx during an action potential triggers SR Ca 2+ release only at subsarcolemmal region. SR Ca 2+ release then spreads towards the central region of the cell thourgh a propagated Ca 2+-induced-Ca 2+ release mechanism. The atrio-ventricular difference in excitation-contraction coupling mechanisms underlies some of the atrioventricular difference in response to physiological and pharmacological stimuli.
Multimodal SHG-2PF Imaging of Microdomain Ca 2+ -Contraction Coupling in Live Cardiac Myocytes
Circulation Research, 2015
Rationale: Cardiac myocyte contraction is caused by Ca 2+ binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca 2+ release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca 2+ release, such as Ca 2+ sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca 2+ -contraction coupling in live cardiac myocytes. Objective: To develop a method for imaging sarcomere level Ca 2+ -contraction coupling in healthy and disease model cardiac myocytes. Methods and Results: Freshly isolated cardiac myocytes were loaded with the Ca 2+ -indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca 2+ signals and sarcomere ...
Journal of Applied Physiology, 2004
Abnormalities in intracellular calcium (Ca 2 i + ) handling have been implicated as the underlying mechanism in a large number of pathologies in the heart. Study into the relation between Ca 2 i + behavior and performance of the whole heart function could provide detailed information into the cellular basis of heart function. In this study we describe a optical ratio imaging set-up and an analysis-method for the beat-to-beat Ca 2 i + video-fluorescence images of an Indo-1 loaded, isolated Tyrode perfused beating rat heart. The signal-to-noise ratio and the spatio-temporal resolution (with an optimum of 1 ms and 0.6 mm respectively) made it possible to register different temporal Ca 2 i + transients together with left ventricle pressure changes. The Ca 2 i + transients showed that Ca 2 i + activation propagates horizontally from left to right during sinus rhythm or from the stimulus-site during direct left ventricle stimulation. The developed Indo-1 ratiometric videotechnique allows the imaging of ratio-changes of Ca 2 i + with a high temporal
Biophysical Journal, 2001
We have developed a distance modulated protocol for scanning ion conductance microscopy to provide a robust and reliable distance control mechanism for imaging contracting cells. The technique can measure rapid changes in cell height from 10 nm to several micrometers, with millisecond time resolution. This has been demonstrated on the extreme case of a contracting cardiac myocyte. By combining this method with laser confocal microscopy, it was possible to simultaneously measure the nanometric motion of the cardiac myocyte, and the local calcium concentration just under the cell membrane. Despite large cellular movement, simultaneous tracking of the changes in cell height and measurement of the intracellular Ca 2ϩ near the cell surface is possible while retaining the cell functionality.
Pulsed laser imaging of rapid Ca2+ gradients in excitable cells
Biophysical Journal, 1994
Excitable cells are thought to respond to action potentials by forming short lived and highly localized Ca2+ gradients near sites of Ca2+ entry or near the site of Ca2+ release by intracellular stores. However, conventional imaging techniques lack the spatial and temporal resolution to capture these gradients. Here we demonstrate the use of pulsed-laser microscopy to measure Ca2+ gradients with submicron spatial resolution and millisecond time resolution in two preparations where the Ca2+ signal is thought to be fast and highly localized: adrenal chromaffin cells, where the entry of Ca2+ through voltage dependent Ca2+ channels triggers exocytotic fusion; and skeletal muscle fibers, where intracellular Ca2+ release from the sarcoplasmic reticulum initiates contraction. In chromaffin cells, Ca2+ gradients developed over 10-100 ms and were initially restricted to discrete submembrane domains, or hot spots, before developing into complete rings of elevated Ca2+ concentration. In frog skeletal muscle large, short-lived (approximately 6 ms) Ca2+ gradients were observed within individual sarcomeres following induction of action potentials. The pulsed laser imaging approach permits, for the first time, the capture and critical examination of rapid Ca2+ signaling events such as those underlying excitation-secretion and excitation-contraction coupling.
AJP: Heart and Circulatory Physiology, 2006
Fluorescence imaging using voltage-sensitive dyes is an important tool for studying electrical propagation in the heart. Yet, the low amplitude of the voltage-sensitive component in the fluorescence signal and high acquisition rates dictated by the rapid propagation of the excitation wave front make it difficult to achieve recordings with high signal-to-noise ratios. Although spatially and temporally filtering the acquired signals has become de facto one of the key elements of optical mapping, there is no consensus regarding their use. Here we characterize the spatiotemporal spectra of optically recorded action potentials and determine the distortion produced by conical filters of different sizes. On the basis of these findings, we formulate the criteria for rational selection of filter characteristics. We studied the evolution of the spatial spectra of the propagating wave front after epicardial point stimulation of the isolated, perfused right ventricular free wall of the pig hear...
Functional cardiac imaging by random access microscopy
Advances in the development of voltage sensitive dyes and Ca 2+ sensors in combination with innovative microscopy techniques allowed researchers to perform functional measurements with an unprecedented spatial and temporal resolution. At the moment, one of the shortcomings of available technologies is their incapability of imaging multiple fast phenomena while controlling the biological determinants involved. In the near future, ultrafast deflectors can be used to rapidly scan laser beams across the sample, performing optical measurements of action potential and Ca 2+ release from multiple sites within cardiac cells and tissues. The same scanning modality could also be used to control local Ca 2+ release and membrane electrical activity by activation of caged compounds and light-gated ion channels. With this approach, local Ca 2+ or voltage perturbations could be induced, simulating arrhythmogenic events, and their impact on physiological cell activity could be explored. The development of this optical methodology will provide fundamental insights in cardiac disease, boosting new therapeutic strategies, and, more generally, it will represent a new approach for the investigation of the physiology of excitable cells.