Figure 1 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Detection and extraction of calcium spikes for analysis. A: a line scan confocal (x-t) image with three indicated regions of interest. The fluorescence intensity is color-coded. The red rectangle delineates the non-cell region used for estimation of the background fluorescence level. The green rectangle delineates the scan region preceding the stimulus (shown above the image) that is used for estimation of baseline cell fluorescence and the level of noise. The white rectangle delineates the region of image chosen for calcium spike detection. The black trace at the left represents the temporal average of fluorescent intensity of the pixels in the white rectangle. The arrowheads mark calcium spikes selected for analysis. B: The time courses of the fluorescent profiles of the spikes marked by the numbered red arrowheads in A. The traces were obtained by averaging 7 spatial pixels delineated by the thin black lines indicated around the calcium spike #2. The time scale is the same for A and B.
Source publication
Intracellular calcium signals are studied by laser-scanning confocal fluorescence microscopy. The required spatio-temporal resolution makes description of calcium signals difficult because of the low signal-to-noise ratio. We designed a new procedure of calcium spike analysis based on their fitting with a model. The accuracy and precision of calciu...
Similar publications
ABSTRACT The magnesium ion, an important constituent of the intracellular milieu in cardiac myocytes, is known,to inhibit ryanodine ,receptor (RyR) Ca, compound ,NP-EGTA. The evoked ,RyR activity was measured ,in planar ,lipid bilayers in the ,presence of 0.6–1.3 mM (free) [Mg 2+ ]at the background,of 3 mM (total) ATP in the presence or absence of...
Catecholaminergic polymorphic ventricular tachycardia (CPVT)is an arrhythmia syndrome characterized by adrenaline induced ventricular tachycardia. The primary genetic aetiologies underlying CPVT are either autosomal dominant or autosomal recessive inheritance, resulting from heterozygous mutations in cardiac ryanodine receptor (RYR2)and homozygous...
![Ca²⁺ waves under different [Ca²⁺]lumen. (A) Computational results for...](publication/324776518/figure/fig5/AS:11431281255799293@1719438645138/Ca-waves-under-different-Calumen-A-Computational-results-for-line-scan-images-of_Q320.jpg)
Ca²⁺ waves in cardiac myocytes can lead to arrhythmias owing to delayed after-depolarisations. Based on Ca²⁺ regulation from the junctional sarcoplasmic reticulum (JSR), a mathematical model was developed to investigate the interplay of clustered and rogue RyRs on Ca²⁺ waves. The model successfully reproduces Ca²⁺ waves in cardiac myocytes, which a...
Citations
... the approach to stabilising the disturbance. To achieve this, a disturbance component was added to Eq. 1, through addition of Gaussian noise [28]. This additional noise affects the Ca 2+ level in the astrocyte cell's cytosol. ...
Synaptic plasticity depends on the gliotransmitters' concentration in the synaptic channel. And, an abnormal concentration of gliotransmitters is linked to neurodegenerative diseases, including Alzheimer's, Parkinson's, and epilepsy. In this paper, a theoretical investigation of the cause of the abnormal concentration of gliotransmitters and how to achieve its control is presented through a Ca2+-signalling-based molecular communications framework. A feed-forward and feedback control technique is used to manipulate IP3 values to stabilise the concentration of Ca2+ inside the astrocytes. The theoretical analysis of the given model aims i) to stabilize the Ca2+ concentration around a particular desired level in order to prevent abnormal gliotransmitters' concentration (extremely high or low concentration can result in neurodegeneration), ii) to improve the molecular communication performance that utilises Ca2+ signalling, and maintain gliotransmitters' regulation remotely. It shows that the refractory periods from Ca2+ can be maintained to lower the noise propagation resulting in smaller time-slots for bit transmission, which can also improve the delay and gain performances. The proposed approach can potentially lead to novel nanomedicine solutions for the treatment of neurodegenerative diseases, where a combination of nanotechnology and gene therapy approaches can be used to elicit the regulated Ca2+ signalling in astrocytes, ultimately improving neuronal activity.
... The existing noise in the florescence Ca 2+ images renders the peak detection process difficult. Although applying smoothing techniques like signal or image filtering may increase signal to noise ratio, it may substantially change the shape and features of Ca 2+ spikes [Balkenius et al., 2015, Janicek et al., 2013 which can distort the experimental results. A common practice for enhancing the performance of peak detection algorithms in noisy data is defining a sliding window with the length of the signal periodicity and identifying global maximum values within the window as signal peaks. ...
... Calcium signal identification and signal source separation are two separate problems in calcium signal analysis. To support the analysis of imaging data many helpful automated or semi-automated computational tools have been developed [18,[21][22][23][24][25][26][27][28]. Therefore, advanced computational tools are helpful to localize, extract and sort cellular signals to detect neuronal calcium activity [18] or to strengthen the detection of calcium activity by signal demixing and denoising [22,23,26]. ...
... The tool can be used as a calcium spike detector, but is, in our opinion, more powerful in evaluating and computing calcium signaling events which are small, irregularly shaped, close to the measurement noise, and in the periphery of the neuron. Different strategies are already used to describe calcium activity automatically and each of the methods has specific advantages [18,[21][22][23]25,45,46]. However, the individual essential requirements on an automated calcium signal detector are quite different, and therefore an all-purpose tool does not exist. ...
... It is a strong feature of our strategy that it works close to the signal noise level and therefore sees signals even if there is an inactive pixel close to an active pixel. On the one hand our strategy sounds risky, as it is prone to overestimate activity events close to the measurement noise [18,23,25] under small SNR values. On the other hand, it does not ignore local activity close to the measurement noise. ...
Local and spontaneous calcium signals play important roles in neurons and neuronal networks. Spontaneous or cell-autonomous calcium signals may be difficult to assess because they appear in an unpredictable spatiotemporal pattern and in very small neuronal loci of axons or dendrites. We developed an open source bioinformatics tool for an unbiased assessment of calcium signals in x,y-t imaging series. The tool bases its algorithm on a continuous wavelet transform-guided peak detection to identify calcium signal candidates. The highly sensitive calcium event definition is based on identification of peaks in 1D data through analysis of a 2D wavelet transform surface. For spatial analysis, the tool uses a grid to separate the x,y-image field in independently analyzed grid windows. A document containing a graphical summary of the data is automatically created and displays the loci of activity for a wide range of signal intensities. Furthermore, the number of activity events is summed up to create an estimated total activity value, which can be used to compare different experimental situations, such as calcium activity before or after an experimental treatment. All traces and data of active loci become documented. The tool can also compute the signal variance in a sliding window to visualize activity-dependent signal fluctuations. We applied the calcium signal detector to monitor activity states of cultured mouse neurons. Our data show that both the total activity value and the variance area created by a sliding window can distinguish experimental manipulations of neuronal activity states. Notably, the tool is powerful enough to compute local calcium events and ‘signal-close-to-noise’ activity in small loci of distal neurites of neurons, which remain during pharmacological blockade of neuronal activity with inhibitors such as tetrodotoxin, to block action potential firing, or inhibitors of ionotropic glutamate receptors. The tool can also offer information about local homeostatic calcium activity events in neurites.
... To analyse the benefit of the feedback control technique with and without feed-forward separately, a noise component is integrated and determine the effectiveness of the approach to stabilising the disturbance. To achieve this, a disturbance component was added to Eq. 1, through addition of Gaussian noise [32]. This additional noise affects the Ca 2+ level in the astrocyte cell's cytosol. ...
Synapses plasticity depends on the gliotransmit-ters' concentration in the synaptic channel. And, an abnormal concentration of gliotransmitters is linked to neurodegenerative diseases, including Alzheimer's, Parkinson's, and Epilepsy. In this paper, a theoretical investigation of the cause of the abnormal concentration of gliotransmitters and how to achieve its control are presented through a Ca 2+-signalling-based molecular communications framework. A feed-forward and feedback control technique is used to manipulate IP3 values to stabilise the concentration of Ca 2+ inside the astrocytes. The theoretical analysis of the given model aims i) to stabilize the Ca 2+ concentration around a particular desired level in order to prevent abnormal gliotransmitters' concentration (extremely high or low concentration can result in neurodegeneration), ii) to improve the molecular communication performance that utilises Ca 2+ signalling, and maintain gliotransmitters' regulation remotely. It shows that the refractory periods from Ca 2+ can be maintained to lower the noise propagation resulting in smaller time-slots for bit transmission, which can also improve the delay and gain performances. The proposed approach can potentially lead to novel nanomedicine solutions for the treatment of neurodegener-ative diseases, where a combination of nanotechnology and gene therapy approaches can be used to elicit the regulated Ca 2+ signalling in astrocytes, ultimately improving neuronal activity.
... In ideal circumstances, such pre-processing is automated, thereby increasing both efficacy and speed of analysis. However, in practical terms, nowadays the abovementioned parameters are still defined by the user or obtained by fitting in most cases [168,169]. ...
Beta cells in the pancreatic islets of Langerhans are precise biological sensors for glucose and play a central role in balancing the organism between catabolic and anabolic needs. A hallmark of the beta cell response to glucose are oscillatory changes of membrane potential that are tightly coupled with oscillatory changes in intracellular calcium concentration which, in turn, elicit oscillations of insulin secretion. Both membrane potential and calcium changes spread from one beta cell to the other in a wave-like manner. In order to assess the properties of the abovementioned responses to physiological and pathological stimuli, the main challenge remains how to effectively measure membrane potential and calcium changes at the same time with high spatial and temporal resolution, and also in as OPEN ACCESS Sensors 2015, 15 27394 many cells as possible. To date, the most widespread approach has employed the electrophysiological patch-clamp method to monitor membrane potential changes. Inherently, this technique has many advantages, such as a direct contact with the cell and a high temporal resolution. However, it allows one to assess information from a single cell only. In some instances, this technique has been used in conjunction with CCD camera-based imaging, offering the opportunity to simultaneously monitor membrane potential and calcium changes, but not in the same cells and not with a reliable cellular or subcellular spatial resolution. Recently, a novel family of highly-sensitive membrane potential reporter dyes in combination with high temporal and spatial confocal calcium imaging allows for simultaneously detecting membrane potential and calcium changes in many cells at a time. Since the signals yielded from both types of reporter dyes are inherently noisy, we have developed complex methods of data denoising that permit for visualization and pixel-wise analysis of signals. Combining the experimental approach of high-resolution imaging with the advanced analysis of noisy data enables novel physiological insights and reassessment of current concepts in unprecedented detail.
... Multiple software applications, particularly in neurobiology, have employed independent component analysis and watershed image segmentation to define individual cells within dense fields and to track region-specific deflections of Ca 2+ -dependent fluorescence (Mukamel et al., 2009;Wong et al., 2010;Watters et al., 2014). Separate automated analysis software has been applied to discern Ca 2+ spiking and oscillation patterns in various cell types, including plant epidermal cells (Russo et al., 2013), cardiac myocytes (Janicek et al., 2013), and T-cells (Salles et al., 2013). The algorithm LC_Pro was recently developed to track the diverse Ca 2+ events in the vascular endothelium (Francis et al., 2012. ...
Although acute and chronic vasoregulation is inherently driven by endothelial Ca²⁺, control and targeting of Ca²⁺-dependent signals are poorly understood. Recent studies have revealed localized and dynamic endothelial Ca²⁺ events comprising an intricate signaling network along the vascular intima. Discrete Ca²⁺ transients emerging from both internal stores and plasmalemmal cation channels couple to specific membrane K⁺ channels, promoting endothelial hyperpolarization and vasodilation. The spatiotemporal tuning of these signals, rather than global Ca²⁺ elevation, appear to direct endothelial functions under physiologic conditions. In fact, altered patterns of dynamic Ca²⁺ signaling may underlie essential endothelial dysfunction in a variety of cardiovascular diseases. Advances in imaging approaches and analyses in recent years have allowed for detailed detection, quantification, and evaluation of Ca²⁺ dynamics in intact endothelium. Here, we discuss recent insights into these signals, including their sources of origination and their functional encoding. We also address key aspects of data acquisition and interpretation, including broad applications of automated high-content analysis.
... We therefore investigated the presence of a repetitive pattern in the sequence of peak-to-peak intervals, as a possible component of the calcium signature. Several techniques of pattern recognition can be found in literature which in most cases analyze the conformity of the experimental data to a predetermined pattern model [46]. Since we had no indication for a precise pattern model that could fit our case, we introduced the calculation of waiting time autocorrelation. ...
Full text here: http://www.biomedcentral.com/content/pdf/1471-2229-13-224.pdf
Repeated oscillations in intracellular calcium (Ca2+) concentration, known as Ca2+ spiking signals, have been described in plants for a limited number of cellular responses to biotic or abiotic stimuli and most notably the common symbiotic signaling pathway (CSSP) which mediates the recognition by their plant hosts of two endosymbiotic microbes, arbuscular mycorrhizal (AM) fungi and nitrogen fixing rhizobia. The detailed analysis of the complexity and variability of the Ca2+ spiking patterns which have been revealed in recent studies requires both extensive datasets and sophisticated statistical tools.
As a contribution, we have developed automated Ca2+ spiking analysis (CaSA) software that performs i) automated peak detection, ii) statistical analyses based on the detected peaks, iii) autocorrelation analysis of peak-to-peak intervals to highlight major traits in the spiking pattern.We have evaluated CaSA in two experimental studies. In the first, CaSA highlighted unpredicted differences in the spiking patterns induced in Medicago truncatula root epidermal cells by exudates of the AM fungus Gigaspora margarita as a function of the phosphate concentration in the growth medium of both host and fungus. In the second study we compared the spiking patterns triggered by either AM fungal or rhizobial symbiotic signals. CaSA revealed the existence of different patterns in signal periodicity, which are thought to contribute to the so-called Ca2+ signature.
We therefore propose CaSA as a useful tool for characterizing oscillatory biological phenomena such as Ca2+ spiking.
... Regarding the image formation, the scanning process of the FIB-SEM images are different from laser scanning confocal microscopes in which, depending on the scanning mode, the pixel acquisition of the next line starts either in a reverse order relative to the preceding line, or in the same order but only after another reversal of the sweep direction [51]. ...
One of the most challenging problems in biological image analysis is the quantification of the dynamical mechanism and complexity of the intracellular space. This paper investigates potential spatial chaos and complex behavior of the intracellular space of typical cancer and normal cell images whose structural details are revealed by the combination of scanning electron microscopy and focused ion beam systems. Such numerical quantifications have important implications for computer modeling and simulation of diseases.Methods
Cancer cell lines derived from a human head and neck squamous cell carcinoma (SCC-61) and normal mouse embryonic fibroblast (MEF) cells produced by focused ion beam scanning electron microscopes were used in this study. Spatial distributions of the organelles of cancer and normal cells can be analyzed at both short range and long range of the bounded dynamical system of the image space, depending on the orientations of the spatial cell. A procedure was designed for calculating the largest Lyapunov exponent, which is an indicator of the potential chaotic behavior in intracellular images. Furthermore, the sample entropy and regularity dimension were applied to measure the complexity of the intracellular images.
Positive values of the largest Lyapunov exponents (LLEs) of the intracellular space of the SCC-61 were obtained in different spatial orientations for both long-range and short-range models, suggesting the chaotic behavior of the cell. The MEF has smaller positive values of LLEs in the long range than those of the SCC-61, and zero vales of the LLEs in the short range analysis, suggesting a non-chaotic behavior. The intracellular space of the SCC-61 is found to be more complex than that of the MEF. The degree of complexity measured in the spatial distribution of the intracellular space in the diagonal direction was found to be approximately twice larger than the complexity measured in the horizontal and vertical directions.
Initial findings are promising for characterizing different types of cells and therefore useful for studying cancer cells in the spatial domain using state-of-the-art imaging technology. The measures of the chaotic behavior and complexity of the spatial cell will help computational biologists gain insights into identifying associations between the oscillation patterns and spatial parameters of cells, and appropriate model for simulating cancer cell signaling networks for cancer treatment and new drug discovery.
Dynamic systems such as cells or tissues generate, either spontaneously or in response to stimuli, transient signals that carry information about the system. Characterization of recorded transients is often hampered by a low signal-to-noise ratio (SNR). Reduction of the noise by filtering has limited use due to partial signal distortion. Occasionally, transients can be approximated by a mathematical function, but such a function may not hold correctly if recording conditions change. We introduce here the model-independent approximation method for general noisy transient signals based on the Gaussian process regression (GPR). The method was implemented in the software TransientAnalyzer, which detects transients in a record, finds their best approximation by the Gaussian process, constructs a surrogate spline function, and estimates specified signal parameters. The method and software were tested on a cellular model of the calcium concentration transient corrupted by various SNR levels and recorded at a low sampling frequency. Statistical analysis of the model data sets provided the error of estimation <7.5% and the coefficient of variation of estimates <17% for peak SNR=5. The performance of GPR on signals of diverse experimental origin was even better than fitting by a function. The software and its description are available on GitHub.
Key points
In cardiac myocytes, subcellular local calcium release signals, calcium sparks, are recruited to form each cellular calcium transient and activate the contractile machinery.
Abnormal timing of recovery of sparks after their termination may contribute to arrhythmias.
We developed a method to interrogate recovery of calcium spark trigger probabilities and their amplitude over time using two‐photon photolysis of a new ultra‐effective caged calcium compound.
The findings confirm the utility of the technique to define an elevated sensitivity of the calcium release mechanism in situ and to follow hastened recovery of spark trigger probabilities in a mouse model of an inherited cardiac arrhythmia, which was used for validation.
Analogous methods are likely to be applicable to investigate other microscopic subcellular signalling systems in a variety of cell types.
Abstract
In cardiac myocytes Ca²⁺‐induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) governs activation of contraction. Ca²⁺ release occurs via subcellular Ca²⁺ signalling events, Ca²⁺ sparks. Local recovery of Ca²⁺ release depends on both SR refilling and restoration of Ca²⁺ sensitivity of the RyRs. We used two‐photon (2P) photolysis of the ultra‐effective caged Ca²⁺ compound BIST‐2EGTA and laser‐scanning confocal Ca²⁺ imaging to probe refractoriness of local Ca²⁺ release in control conditions and in the presence of cAMP or low‐dose caffeine (to stimulate CICR) or cyclopiazonic acid (CPA; to slow SR refilling). Permeabilized cardiomyocytes were loaded with BIST‐2EGTA and rhod‐2. Pairs of short 2P photolytic pulses (1 ms, 810 nm) were applied with different intervals to test Ca²⁺ release amplitude recovery and trigger probability for the second spark in a pair. Photolytic and biological events were distinguished by classification with a self‐learning support vector machine (SVM) algorithm. In permeabilized myocytes data recorded in the presence of CPA showed a lower probability of triggering a second spark compared to control or cAMP conditions. Cardiomyocytes from a mouse model harbouring the arrhythmogenic RyRR420Q mutation were used for further validation and revealed a higher Ca²⁺ sensitivity of CICR. This new 2P approach provides composite information of Ca²⁺ release amplitude and trigger probability recovery reflecting both SR refilling and restoration of CICR and RyR Ca²⁺ sensitivity. It can be used to measure the kinetics of local CICR recovery, alterations of which may be related to premature heart beats and arrhythmias.
