A new approach to the detection and statistical classification of Ca2+ sparks

The availability of high-speed, two-dimensional (2-D) confocal microscopes and the expanding armamentarium of fluorescent probes presents unprecedented opportunities and new challenges for studying the spatial and temporal dynamics of cellular processes. The need to remove subjectivity from the detection process, the difficulty of the human eye to detect subtle changes in fluorescence in these 2-D images, and the large volume of data produced by these confocal microscopes call for the need to develop algorithms to automatically mark the changes in fluorescence. These fluorescence signal changes are often subtle, so the statistical estimate of the likelihood that the detected signal is not noise is an integral part of the detection algorithm. This statistical estimation is fundamental to our new approach to detection; in earlier Ca(2+) spark detectors, this statistical assessment was incidental to detection. Importantly, the use of the statistical properties of the signal local to the spark, instead of over the whole image, reduces the false positive and false negative rates. We developed an automatic spark detection algorithm based on these principles and used it to detect sparks on an inhomogeneous background of transverse tubule-labeled rat ventricular cells. Because of the large region of the cell surveyed by the confocal microscope, we can detect a large enough number of sparks to measure the dynamic changes in spark frequency in individual cells. We also found, in contrast to earlier results, that cardiac sparks are spatially symmetric. This new approach puts the detection of fluorescent signals on a firm statistical foundation.     

Automated Detection and Analysis of Ca Sparks in x-y Image Stacks Using a Thresholding Algorithm Implemented within the Open-Source Image Analysis Platform ImageJ

Previous studies have used analysis of Ca²⁺ sparks extensively to investigate both normal and pathological Ca²⁺ regulation in cardiac myocytes. The great majority of these studies used line-scan confocal imaging. In part, this is because the development of open-source software for automatic detection of Ca²⁺ sparks in line-scan images has greatly simplified data analysis. A disadvantage of line-scan imaging is that data are collected from a single row of pixels, representing only a small fraction of the cell, and in many instances x-y confocal imaging is preferable. However, the limited availability of software for Ca²⁺ spark analysis in two-dimensional x-y image stacks presents an obstacle to its wider application. This study describes the development and characterization of software to enable automatic detection and analysis of Ca²⁺ sparks within x-y image stacks, implemented as a plugin within the open-source image analysis platform ImageJ. The program includes methods to enable precise identification of cells within confocal fluorescence images, compensation for changes in background fluorescence, and options that allow exclusion of events based on spatial characteristics.     

Automated Detection and Analysis of Ca2+ Sparks in x-y Image Stacks Using a Thresholding Algorithm Implemented within the Open-Source Image Analysis Platform ImageJ

Previous studies have used analysis of Ca²⁺ sparks extensively to investigate both normal and pathological Ca²⁺ regulation in cardiac myocytes. The great majority of these studies used line-scan confocal imaging. In part, this is because the development of open-source software for automatic detection of Ca²⁺ sparks in line-scan images has greatly simplified data analysis. A disadvantage of line-scan imaging is that data are collected from a single row of pixels, representing only a small fraction of the cell, and in many instances x-y confocal imaging is preferable. However, the limited availability of software for Ca²⁺ spark analysis in two-dimensional x-y image stacks presents an obstacle to its wider application. This study describes the development and characterization of software to enable automatic detection and analysis of Ca²⁺ sparks within x-y image stacks, implemented as a plugin within the open-source image analysis platform ImageJ. The program includes methods to enable precise identification of cells within confocal fluorescence images, compensation for changes in background fluorescence, and options that allow exclusion of events based on spatial characteristics.     

SparkMaster: automated calcium spark analysis with ImageJ

Ca sparks are elementary Ca-release events from intracellular Ca stores that are observed in virtually all types of muscle. Typically, Ca sparks are measured in the line-scan mode with confocal laser-scanning microscopes, yielding two-dimensional images (distance vs. time). The manual analysis of these images is time consuming and prone to errors as well as investigator bias. Therefore, we developed SparkMaster, an automated analysis program that allows rapid and reliable spark analysis. The underlying analysis algorithm is adapted from the threshold-based standard method of spark analysis developed by Cheng et al. (Biophys J 76: 606–617, 1999) and is implemented here in the freely available image-processing software ImageJ. SparkMaster offers a graphical user interface through which all analysis parameters and output options are selected. The analysis includes general image parameters (number of detected sparks, spark frequency) and individual spark parameters (amplitude, full width at half-maximum amplitude, full duration at half-maximum amplitude, full width, full duration, time to peak, maximum steepness of spark upstroke, time constant of spark decay). We validated the algorithm using images with synthetic sparks embedded into backgrounds with different signal-to-noise ratios to determine an analysis criteria at which a high sensitivity is combined with a low frequency of false-positive detections. Finally, we applied SparkMaster to analyze experimental data of sparks measured in intact and permeabilized ventricular cardiomyocytes, permeabilized mammalian skeletal muscle, and intact smooth muscle cells. We found that SparkMaster provides a reliable, easy to use, and fast way of analyzing Ca sparks in a wide variety of experimental conditions. myocytes; sarcoplasmic reticulum; confocal microscopy     

Smooth muscle cells and interstitial cells of blood vessels

A rise in intracellular ionised calcium concentration ([Ca(2+)](i)) at sites adjacent to the contractile proteins is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques with special accent on the fluorescence confocal microscopy and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for study of the relationship between the structural organisation of the living smooth muscle myocyte and the features of calcium signalling at subcellular level. Applying fluorescent confocal microscopy and tight-seal recording of transmembrane ion currents to freshly isolated vascular myocytes we have demonstrated that: (1) Ca(2+) sparks originate from clustered opening of ryanodine receptors (RyRs) and build up a cell-wide increase in [Ca(2+)](i) upon myocyte excitation; (2) spontaneous Ca(2+) sparks occurred at the highest rate at certain preferred locations, frequent discharge sites (FDS), which are associated with a prominent portion of the sarcoplasmic reticulum (SR) located close to the cell membrane; (3) Ca(2+)-dependent K(+) and Cl(-) channels sense the local changes in [Ca(2+)](i) during a calcium spark and thereby couple changes in [Ca(2+)](i) within a microdomain to changes in the membrane potential, thus affecting excitability of the cell; (4) an intercommunication between RyRs and inositol trisphosphate receptors (IP(3)Rs) is one of the important determinants of intracellular calcium dynamics that, in turn, can modulate the cell membrane potential through differential targeting of calcium dependent membrane ion channels. Furthermore, using immunohystochemical approaches in combination with confocal imaging we identified non-contractile cells closely resembling interstitial cells (ICs) of Cajal (which are considered to be pacemaker cells in the gut) in the wall of portal vein and mesenteric artery. Using electron microscopy, tight-seal recording and fluorescence confocal imaging we obtained information on the morphology of ICs and their possible coupling to smooth muscle cells (SMCs), calcium signalling in ICs and their electrophysiological properties. The functions of these cells are not yet fully understood; in portal vein they may act as pacemakers driving the spontaneous activity of the muscle; in artery they may have other a yet unsuspected functions.     

Life and death of a cardiac calcium spark

Calcium sparks in cardiac myocytes are brief, localized calcium releases from the sarcoplasmic reticulum (SR) believed to be caused by locally regenerative calcium-induced calcium release (CICR) via couplons, clusters of ryanodine receptors (RyRs). How such regeneration is terminated is uncertain. We performed numerical simulations of an idealized stochastic model of spark production, assuming a RyR gating scheme with only two states (open and closed). Local depletion of calcium in the SR was inevitable during a spark, and this could terminate sparks by interrupting CICR, with or without assumed modulation of RyR gating by SR lumenal calcium. Spark termination by local SR depletion was not robust: under some conditions, sparks could be greatly and variably prolonged, terminating by stochastic attrition–a phenomenon we dub “spark metastability.” Spark fluorescence rise time was not a good surrogate for the duration of calcium release. Using a highly simplified, deterministic model of the dynamics of a couplon, we show that spark metastability depends on the kinetic relationship of RyR gating and junctional SR refilling rates. The conditions for spark metastability resemble those produced by known mutations of RyR2 and CASQ2 that cause life-threatening triggered arrhythmias, and spark metastability may be mitigated by altering the kinetics of the RyR in a manner similar to the effects of drugs known to prevent those arrhythmias. The model was unable to explain the distributions of spark amplitudes and rise times seen in chemically skinned cat atrial myocytes, suggesting that such sparks may be more complex events involving heterogeneity of couplons or local propagation among sub-clusters of RyRs.     

Amplitude distribution of calcium sparks in confocal images: theory and studies with an automatic detection method.

Determination of the calcium spark amplitude distribution is of critical importance for understanding the nature of elementary calcium release events in striated muscle. In the present study we show, on general theoretical grounds, that calcium sparks, as observed in confocal line scan images, should have a nonmodal, monotonic decreasing amplitude distribution, regardless of whether the underlying events are stereotyped. To test this prediction we developed, implemented, and verified an automated computer algorithm for objective detection and measurement of calcium sparks in raw image data. When the sensitivity and reliability of the algorithm were set appropriately, we observed highly left-skewed or monotonic decreasing amplitude distributions in skeletal muscle cells and cardiomyocytes, confirming the theoretical predictions. The previously reported modal or Gaussian distributions of sparks detected by eye must therefore be the result of subjective detection bias against small amplitude events. In addition, we discuss possible situations when a modal distribution might be observed.     

Increasing sensitivity of Ca2+ spark detection in noisy images by application of a matched-filter object detection algorithm

Microscopic calcium (Ca²⁺) events (such as Ca²⁺ sparks) are an important area for study, as they help clarify the mechanism(s) underlying intracellular signaling. In the heart, Ca²⁺ sparks occur as a result of Ca²⁺ release from the sarcoendoplasmic reticulum, via ryanodine receptor channels. Measurement of Ca²⁺ spark properties can provide valuable information about the control of ryanodine receptor channel gating in situ, but requires high spatiotemporal resolution imaging, which produces noisy datasets that are problematic for spark detection. Automated detection algorithms may overcome visual detection bias, but missed and false-positive events can distort the distribution of measured Ca²⁺ spark properties. We present a sensitive and reliable method for the automated detection of Ca²⁺ sparks in datasets obtained using confocal line-scanning or total internal reflection fluorescence microscopy. This matched-filter detection algorithm (MFDA) employs a user-defined object, chosen to mimic Ca²⁺ spark properties, and the experimental dataset is searched for instances of the object. Detection certainty is provided by nonparametric statistical testing. The supplied codes can also refine the search object on the basis of those detected to further increase detection sensitivity. In comparison to a commonly used, intensity-thresholding algorithm, the MFDA is more sensitive and reliable, particularly at low signal/noise ratios. The MFDA can also be easily adapted to other signal-detection problems in noisy datasets.     

A preferred amplitude of calcium sparks in skeletal muscle

In skeletal and cardiac muscle, calcium release from the sarcoplasmic reticulum, leading to contraction, often results in calcium sparks. Because sparks are recorded by confocal microscopy in line-scanning mode, their measured amplitude depends on their true amplitude and the position of the spark relative to the scanned line. We present a method to derive from measured amplitude histograms the actual distribution of spark amplitudes. The method worked well when tested on simulated distributions of experimental sparks. Applied to massive numbers of sparks imaged in frog skeletal muscle under voltage clamp in reference conditions, the method yielded either a decaying amplitude distribution (6 cells) or one with a central mode (5 cells). Caffeine at 0.5 or 1 mM reversibly enhanced this mode (5 cells) or induced its appearance (4 cells). The occurrence of a mode in the amplitude distribution was highly correlated with the presence of a mode in the distribution of spark rise times or in the joint distribution of rise times and spatial widths. If sparks were produced by individual Markovian release channels evolving reversibly, they should not have a preferred rise time or amplitude. Channel groups, instead, could cooperate allosterically or through their calcium sensitivity, and give rise to a stereotyped amplitude in their collective spark.     

Effects of ryanoids on spontaneous and depolarization-evoked calcium release events in frog muscle

The effects of ryanoids on calcium sparks and transients were studied in voltage-clamped cut frog muscle fibers with a laser scanning confocal microscope. For each ryanoid employed, several sequential effects were observed, including: a), transient increases in spontaneous spark frequency; b), conversions of sparks to long-lasting steady glows; and c), occasional interruptions of the glows. The ratio of the amplitude of the glow induced by a ryanoid to that of the precursory spark followed the order: ryanodol > ryanodine > C(10)-O(eq)-glycyl-ryanodine > C(10)-O(eq)-beta-alanyl-ryanodol. This sequence of glow amplitudes parallels that of the subconductances induced by these ryanoids in single-channel studies, suggesting that the glows reflect Ca(2+) fluxes through semiopen calcium release channels. Ryanoids also abolished depolarization-evoked sparks elicited with small pulses, and transformed the calcium release during depolarization to a uniform nonsparking fluorescence signal. The ratio of this signal, averaged spatially, to that of the control followed the order: ryanodol < ryanodine < C(10)-O(eq)-glycyl-ryanodine < C(10)-O(eq)-beta-alanyl-ryanodol, implying an inverse relationship with the amplitudes of ryanoid-induced glows. The observation that depolarization-evoked calcium release can occur after ryanoid suppression of calcium sparks suggests the possibility of a new strategic approach for treating skeletal muscle diseases resulting from leaky calcium release channels.     

Nonlinear propagation of spherical calcium waves in rat cardiac myocytes.

Spontaneous calcium waves in enzymatically isolated rat cardiac myocytes were investigated by confocal laser scanning microscopy (CLSM) using the fluorescent Ca2+-indicator fluo-3 AM. As recently shown, a spreading wave of enhanced cytosolic calcium appears, most probably during Ca2+ overload, and is initiated by an elementary event called a "calcium spark." When measured by conventional fluorescence microscopy the propagation velocity of spontaneous calcium waves determined at several points along the cardiac myocyte was previously found to be constant. More precise measurements with a CLSM showed a nonlinear propagation. The wave velocity was low, close to the focus, and increased with increasing time and propagation length, approaching a maximum of 113 microns/s. This result was surprising, inasmuch as for geometrical reasons a decrease of the propagation velocity might be expected if the confocal plane is not identical with that plane where the focus of the wave was localized. It is suggested that the propagation velocity is essentially dependent on the curvature of the spreading wave. From the linear relationship of velocity versus curvature, a critical radius of 2.7 +/- 1.4 microns (mean +/- SD) was worked out, below which an outward propagation of the wave will not take place. Once released from a sufficiently extended cluster of sarcoplasmic reticulum release channels, calcium diffuses and will activate its neighbors. While traveling away, the volume into which calcium diffuses becomes effectively smaller than at low radii. This effect is the consequence of the summation of elementary events (Ca2+ sparks) and leads to a steeper increase of the cytosolic calcium concentration after a certain diffusion path length. Thus the time taken to reach a critical threshold of [Ca2+]i at the neighboring calcium release sites decreases with decreasing curvature and the wave will propagate faster.     

Calcium alternans in a couplon network model of ventricular myocytes: role of sarcoplasmic reticulum load

Intracellular calcium (Ca) alternans in cardiac myocytes have been shown in many experimental studies, and the mechanisms remain incompletely understood. We recently developed a "3R theory" that links Ca sparks to whole cell Ca alternans through three critical properties: randomness of Ca sparks; recruitment of a Ca spark by neighboring Ca sparks; and refractoriness of Ca release units. In this study, we used computer simulation of a physiologically detailed mathematical model of a ventricular myocyte couplon network to study how sarcoplasmic reticulum (SR) Ca load and other physiological parameters, such as ryanodine receptor sensitivity, SR uptake rate, Na-Ca exchange strength, and Ca buffer levels affect Ca alternans in the context of 3R theory. We developed a method to calculate the parameters used in the 3R theory (i.e., the primary spark rate and the recruitment rate) from the physiologically detailed Ca cycling model and paced the model periodically to elicit Ca alternans. We show that alternans only occurs for an intermediate range of the SR Ca load, and the underlying mechanism can be explained via its effects on the 3Rs. Furthermore, we show that altering the physiological parameters not only directly changes the 3Rs but also alters the SR Ca load, having an indirect effect on the 3Rs as well. Therefore, our present study links the SR Ca load and other physiological parameters to whole cell Ca alternans through the framework of the 3R theory, providing a general mechanistic understanding of Ca alternans in ventricular myocytes.     

Time Course of Individual Ca2+ Sparks in Frog Skeletal Muscle Recorded at High Time Resolution

Discrete Ca²⁺ release events (Ca²⁺ “sparks”) were recorded in cut segments of single frog skeletal muscle fibers using a video-rate laser-scanning confocal microscope operating in line-scan mode (63 μs per line). Fibers loaded with the Ca²⁺ indicator fluo-3 were voltage clamped at a holding potential of 0 mV, briefly reprimed at −90 mV, and then strongly depolarized with a large test pulse to activate any reprimed voltage sensors. Using this high time resolution system, it was possible to record individual Ca²⁺ sparks at ∼30-fold higher time resolution than previously attained. The resulting new experimental data provides a means of characterizing the time course of fluorescence during the brief (a few milliseconds) rising phase of a spark, which was not possible with the previously used 1.5–2 ms per line confocal systems. Analysis of the time course of individual identified events indicates that fluorescence begins to rise rather abruptly at the start of the spark, continues to rise at a slightly decreasing rate to a relatively sharp peak, and then declines along a quasi-exponential time course. The mean rise time of 198 sparks was 4.7 ± 0.1 ms, and there was no correlation between rise time and peak amplitude. Average sparks constructed by temporally and spatially superimposing and summing groups of individual sparks having similar rise times gave a lower noise representation of the sparks, consistent with the time course of individual events. In theory, the rising phase of a spark provides a lower bound estimation of the time that Ca²⁺ ions are being released by the sarcoplasmic reticulum Ca²⁺ channel(s) generating the spark. The observed time course of fluorescence suggests that the Ca²⁺ release underlying a spark could continue at a fairly constant rate throughout the rising phase of the spark, and then stop rather abruptly at the time of the peak.     

Modulation of local Ca2+ release sites by rapid fluid puffing in rat atrial myocytes

Atrial myocytes that lack t-tubules appear to have two functionally separate sarcoplasmic Ca2+ stores: a peripheral store associated with plasmalemmal L-type calcium channels and a central store with no apparent proximity to L-type calcium channels. Here we describe a set of calcium sparks and waves that are triggered by puffing of pressurized (200-400 mmH2O) bathing solutions onto resting isolated rat atrial myocytes. Puffing of pressurized (200 mmH2O) solutions, identical to those bathing the myocytes from distances of approximately 150 microm onto the surface of a single myocyte triggered or enhanced spontaneously occurring peripheral sparks by five- to six-fold and central Ca2+ sparks by two- to three-fold, without altering the unitary spark properties. Exposure to higher pressure flows (400 mmH2O) often triggered longitudinally spreading Ca2+ waves. These results suggest that pressurized flows may directly modulate Ca2+ signaling of atrial myocytes by activating the intracellular Ca2+ release sites.     

A mathematical analysis of the generation and termination of calcium sparks

Calcium sparks are local regenerative releases of Ca(2+) from a cluster of ryanodine receptors on the sarcoplasmic reticulum. During excitation-contraction coupling in cardiac cells, Ca(2+) sparks are triggered by Ca(2+) entering the cell via the T-tubules (Ca(2+)-induced Ca(2+) release). However under conditions of calcium overload, Ca(2+) sparks can be triggered spontaneously. The exact process by which Ca(2+) sparks terminate is still an open question, although both deterministic and stochastic processes are likely to be important. In this article, asymptotic methods are used to analyze a single Ca(2+) spark model, which includes both deterministic and stochastic biophysical mechanisms. The analysis calculates both spark frequencies and spark duration distributions, and shows under what circumstances stochastic transitions are important. Additionally, a model of the coupling of the release channels via the FK-binding protein is analyzed.     

Direct visualization of sarcoplasmic reticulum regions discharging Ca(2+)sparks in vascular myocytes

Localized Ca(2+)-release events, Ca(2+)sparks, have been suggested to be the 'elementary building blocks' of the calcium signalling system in all types of muscles. In striated muscles these occur at regular intervals along the fibre corresponding to the sarcomeric structures which do not exist in smooth muscle. We showed previously that in visceral and vascular myocytes Ca(2+)sparks occurred much more frequently at certain sites (frequent discharge sites [FDSs]). In this paper, we have related the position of FDSs to the distribution of the sarcoplasmic reticulum in the same living myocyte. The three-dimensional distribution of the SR in freshly isolated rabbit portal vein myocytes was visualized by means of high-resolution confocal imaging after staining with DiOC(6)and/or BODIPY TR-X ryanodine. Both fluorochromes revealed a similar staining pattern indicating a helical arrangement of well-developed superficial SR which occupied about 6% of the cell volume. Computing the frequency of spontaneous Ca(2+)sparks detected by means of fluo-4 fluorescence revealed that in about 70% of myocytes there was only one major FDS located on a prominent portion of superficial SR network usually within 1-2 microm of the nuclear envelope, although a few sparks occurred at other sites scattered generally in superficial locations throughout the cell. Polarized mitochondria were readily identified by accumulation of tetramethylrhodamine ethyl ester (TMRE). These were closely associated with the SR network in extra-nuclear regions. TMRE staining, however, failed to reveal any mitochondria near the FDS-related SR element. When observed, propagating [Ca(2+)](i)waves and associated myocyte contractions were initiated at FDSs. This study provide first insight into the three-dimensional arrangement of the SR in living smooth muscle cells and relates the peculiarity of the structural organization of the myocyte to the features of Ca(2+)signalling at subcellular level.     

Detecting Ca2+ sparks on stationary and varying baselines

Studies concerning the physiological significance of Ca(2+) sparks often depend on the detection and measurement of large populations of events in noisy microscopy images. Automated detection methods have been developed to quickly and objectively distinguish potential sparks from noise artifacts. However, previously described algorithms are not suited to the reliable detection of sparks in images where the local baseline fluorescence and noise properties can vary significantly, and risk introducing additional bias when applied to such data sets. Here, we describe a new, conceptually straightforward approach to spark detection in linescans that addresses this issue by combining variance stabilization with local baseline subtraction. We also show that in addition to greatly increasing the range of images in which sparks can be automatically detected, the use of a more accurate noise model enables our algorithm to achieve similar detection sensitivities with fewer false positives than previous approaches when applied both to synthetic and experimental data sets. We propose, therefore, that it might be a useful tool for improving the reliability and objectivity of spark analysis in general, and describe how it might be further optimized for specific applications.     

Improved spark and ember detection using stationary wavelet transforms

Calcium sparks and embers are localized intracellular events of calcium release in muscle cells studied frequently by confocal microscopy using line-scan imaging. The large quantity of images and large number of events require automatic detection procedures based on signal processing methods. In the past decades these methods were based on thresholding procedures. Although, recently, wavelet transforms were also introduced, they have not become widespread. We have implemented a set of algorithms based on one- and two-dimensional versions of the à trous wavelet transform. The algorithms were used to perform spike filtering, denoising and detection procedures. Due to the dependence of the algorithms on user adjustable parameters, their effect on the efficiency of the algorithm was studied in detail. We give methods to avoid false positive detections which are the consequence of the background noise in confocal images. In order to establish the efficiency and reliability of the algorithms, various tests were performed on artificial and experimental images. Spark parameters (amplitude, full width-at-half maximum) calculated using the traditional and the wavelet methods were compared. We found that the latter method is capable of identifying more events with better accuracy on experimental images. Furthermore, we extended the wavelet-based transform from calcium sparks to long-lasting small-amplitude events as calcium embers. The method not only solved their automatic detection but enabled the identification of events with small amplitude that otherwise escaped the eye, rendering the determination of their characteristic parameters more accurate.     

Effect of endogenous and exogenous nitric oxide on calcium sparks as targets for vasodilation in rat cerebral artery

The potent vasodilator nitric oxide (NO), produced mainly by the endothelium, acts through a BK_Ca-dependent mechanism to increase the frequency of calcium sparks (Ca²⁺ sparks) in myocyte isolated from rat cerebral arteries. Our present aim has been to assess the role of endogenous and exogenous NO on the Ca²⁺ sparks through ryanodine-sensitive channels in the sarcoplasmic reticulum of an intact artery. Calcium sparks, detected with fluo-4 and laser scanning confocal microscopy, were examined in isolated pressurized rat posterior cerebral arteries with (intact) and without endothelium (denuded). Addition of the NO donor, DEA-NONOate (N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine), did not change the amplitude and frequency of Ca²⁺ sparks in the intact artery. However, inhibition of nitric oxide synthase with N-ω-nitro-l-arginine or removal of endothelium reduced Ca²⁺ sparks frequency by about 50%. Under these conditions (i.e., absence of endogenous NO production), DEA-NONOate, increased Ca²⁺ spark frequency 3- to 4-fold. These results suggest that endothelial NO modulates local Ca²⁺ release events in the arterial smooth muscle and that this mechanism may contribute to the actions of nitrovasodilators.     

Frequency division multiplexed multichannel high-speed fluorescence confocal microscope

In this article, we report a new type of fluorescence confocal microscope: frequency division multiplexed multichannel fluorescence confocal microscope, in which we encode the spatial location information into the frequency domain. In this microscope, the exciting laser beam is first split into multiple beams and each beam is modulated at a different frequency. These multiple beams are focused at different locations of the target to form multiple focal points, which further generate multiple fluorescent emission spots. The fluorescent emissions from different focal points are also modulated at different frequencies, because the exciting beams are modulated at different frequencies (or difference carrier frequency). Then, all the fluorescent emissions (modulated at different frequencies) are collected together and detected by a highly sensitive, large-dynamic-range photomultiplier tube. By demodulating the detected signal (i.e., via the Fourier transform), we can distinguish the fluorescent light emitted from the different locations by the corresponding carrier frequencies. The major advantage of this unique fluorescence confocal microscope is that it not only has a high sensitivity because of the use of photomultiplier tube but also can get multiple-point data simultaneously, which is crucial to study the dynamic behavior of many biological process. As an initial step, to verify the feasibility of the proposed multichannel confocal microscope, we have developed a two-channel confocal fluorescence microscope and applied it to study the dynamic behavior of the changes of the calcium ion concentration during the single cardiac myocyte contraction. Our preliminary experimental results demonstrated that we could indeed realize multichannel confocal fluorescence microscopy by utilizing the frequency division multiplexed microscope, which could become an effective tool to study the dynamic behavior of many biological processes.