Intracellular Calcium Dynamics Research Articles

Experimental and Mathematical Modeling of Intracellular Calcium Dynamics for Anticancer Effects Evaluation in Esophageal Cancer

Targeting dysregulated Ca2+ signaling in cancer cells is an emerging chemotherapy approach. This group previously reported that store‐operated Ca2+ entry (SOCE) blocker, such as RP4010, could effectively inhibit intracellular Ca2+ oscillations and cell proliferation in esophageal cancer, a leading cancer worldwide with low 5‐year survival rate. While many current clinical chemotherapy drugs, such as tyrosine kinase inhibitors (TKIs) can induce intracellular Ca2+ release, it is not known whether combination of SOCE blockers and TKIs could achieve better chemotherapy effects. The present study employed both experimental and mathematical models to evaluate the effect of RP4010 and TKI (Afatinib) on intracellular Ca2+ oscillation periods in KYSE‐150 cells, an esophageal cancer cell line. The intracellular Ca2+ oscillation period in KYSE‐150 cells was measured as ~32.8 sec; both Afatinib and RP4010 could reduce intracellular Ca2+ and prolong the period in a dose‐dependent manner. Using a modified canonical model with variables of SOCE channel and IP3 receptor affected by RP4010 and Afatinib receptively, the intracellular Ca2+ oscillation dynamics were simulated. The theoretical data of the combined implementations of drugs also fitted well with experimental data. Both results demonstrated a synergetic effect of RP4010 and Afatinib on inhibiting intracellular Ca2+ oscillation period, which was further confirmed by cell proliferation analysis. This study suggests that experimental and mathematical modeling of intracellular Ca2+ dynamics could be used as a rapid and cost‐effective tool for evaluation of combined chemotherapy drugs.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Local Anesthetics Induce Cardiac Toxicity Through Distinct Effects on Calcium Dynamics: Implications For Clinical Prevention

INTRODUCTIONThe clinically safe dose of local anesthetics is limited by their induction of potentially lethal cardiac arrhythmias, which occurs in ~50,000 people in the US per year. While this drug toxicity is often attributed to non‐specific block of cardiac sodium channels, studies on isolated channels fail to show any clinically relevant difference between more harmful (i.e. bupivacaine, BUP) and less harmful (i.e. ropivacaine, ROP) anesthetics. We sought to determine the mechanisms of toxicity and arrhythmogenesis for BUP and ROP, revealing prevention and treatment strategies to improve clinical safety.METHODSWe cultured beating 2D‐tissues of human iPSC‐derived cardiomyocytes and treated them with clinically relevant concentrations of BUP or ROP. We then analyzed changes to ion and contractile dynamics using the xCELLigence RTCA Cardio and the cytosolic calcium dye FLIPR5. As an inotrope, calcium was tested for its ability to recover contractile dysfunction. Our in vitro findings were validated in a rat model of BUP toxicity. Anesthetized animals were infused with either saline or CaCl2 (10 mg/kg over 5 min) in the tail vein, followed by either 2 mg/kg/min BUP or ROP, until asystole. Cardiac activity was monitored via ECG and invasive arterial blood pressure recordings, and analysed using non‐linear regression incorporating the effects of drug treatment, sampling time, and animal variability.RESULTSIn human iPSC cardiomyocytes, both anesthetics affected contractility, beat rate, rhythm, excitation‐contraction coupling, and sodium channel activity as expected, with BUP causing a greater adverse effect. BUP, but not ROP, additionally delayed calcium wave duration by 20% and reduced overall calcium flux by 66%. Interestingly, CaCl2 co‐treatment with BUP abolished aberrancy in all toxicity metrics, where CaCl2 co‐treatment with ROP exacerbated toxicity. Further investigation into intracellular calcium dynamics revealed BUP‐specific defects in diastolic and systolic calcium flux, which were mitigated by CaCl2 co‐treatment. Our in vivo model validated these findings, with ROP showing less toxicity than BUP in all observed metrics. CaCl2 pre‐treatment mitigated the BUP‐mediated decline in beat rate and diastolic arterial pressure, but had no effect, or exacerbated outcomes, in ROP‐treated rats. Furthermore, CaCl2 pre‐treatment reduced arrhythmia onset and QRS widening cased by BUP, but exacerbated these arrhythmia metrics and reduced survival with ROP.CONCLUSIONSWe found that BUP, but not ROP, caused a baseline dysfunction in calcium dynamics in human iPSC‐derived cardiomyocytes. Calcium pre‐treatment prevented BUP, but exacerbated ROP cardiotoxicity in both in vitro and in vivo models. Together, our results implicate distinct calcium signaling and dynamics in eliciting the higher clinical cardiotoxicity of the BUP local anesthetic, and show how calcium can be used to protect against the arrhythmogenic effects of BUP overdose.Support or Funding InformationCanadian Institutes of Health ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multimodal on-axis platform for all-optical electrophysiology with near-infrared probes in human stem-cell-derived cardiomyocytes

Combined optogenetic stimulation and optical imaging permit scalable, contact-free high-throughput probing of cellular electrophysiology and optimization of stem-cell derived excitable cells, such as neurons and muscle cells. We report a new “on-axis” configuration (combined single optical path for stimulation and for multiparameter imaging) of OptoDyCE, our all-optical platform for studying human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) and other cell types, optically driven by Channelrhodopsin2 (ChR2). This solid-state system integrates optogenetic stimulation with temporally-multiplexed simultaneous recording of membrane voltage (Vm) and intracellular calcium ([Ca2+]i) dynamics using a single photodetector. We demonstrate the capacity for combining multiple spectrally-compatible actuators and sensors, including newer high-performance near-infrared (NIR) voltage probes BeRST1 and Di-4-ANBDQBS, to record complex spatiotemporal responses of hiPSC-CMs to drugs in a high-throughput manner.

Open-Source Multiparametric Optocardiography

Since the 1970s fluorescence imaging has become a leading tool in the discovery of mechanisms of cardiac function and arrhythmias. Gradual improvements in fluorescent probes and multi-camera technology have increased the power of optical mapping and made a major impact on the field of cardiac electrophysiology. Tandem-lens optical mapping systems facilitated simultaneous recording of multiple parameters characterizing cardiac function. However, high cost and technological complexity restricted its proliferation to the wider biological community. We present here, an open-source solution for multiple-camera tandem-lens optical systems for multiparametric mapping of transmembrane potential, intracellular calcium dynamics and other parameters in intact mouse hearts and in rat heart slices. This 3D-printable hardware and Matlab-based RHYTHM 1.2 analysis software are distributed under an MIT open-source license. Rapid prototyping permits the development of inexpensive, customized systems with broad functionality, allowing wider application of this technology outside biomedical engineering laboratories.

Retinal Characterization of the Thy1-GCaMP3 Transgenic Mouse Line After Optic Nerve Transection.

GCaMP3 is a genetically encoded calcium indicator for monitoring intracellular calcium dynamics. We characterized the expression pattern and functional properties of GCaMP3 in the Thy1-GCaMP3 transgenic mouse retina. To determine the specificity of GCaMP3 expression, Thy1-GCaMP3 (B6; CBA-Tg(Thy1-GCaMP3)6Gfng/J) retinas were processed for immunohistochemistry with anti-green fluorescent protein (anti-GFP, to enhance GCaMP3 fluorescence), anti-RBPMS (retinal ganglion cell [RGC]-specific marker), and antibodies against amacrine cell markers (ChAT, GABA, GAD67, syntaxin). Calcium imaging was used to characterize functional responses of GCaMP3-expressing (GCaMP+) cells by recording calcium transients evoked by superfusion of kainic acid (KA; 10, 50, or 100 μM). In a subset of animals, optic nerve transection (ONT) was performed 3, 5, or 7 days prior to calcium imaging. GFP immunoreactivity colocalized with RBPMS but not amacrine cell markers in both ONT and non-ONT (control) groups. Calcium transients evoked by KA were reduced after ONT (50 μM KA; ΔF/F0 [SD]; control: 1.00 [0.67], day 3: 0.50 [0.35], day 5: 0.31 [0.28], day 7: 0.35 [0.36]; P < 0.05 versus control). There was also a decrease in the number of GCaMP3+ cells after ONT (cells/mm2 [SD]; control: 2198 [453], day 3: 2224 [643], day 5: 1383 [375], day 7: 913 [178]; P < 0.05). Furthermore, the proportion of GCaMP3+ cells that responded to KA decreased after ONT (50 μM KA, 97%, 54%, 47%, and 58%; control, 3, 5, and 7 days, respectively). Following ONT, functional RGC responses are lost prior to the loss of RGC somata, suggesting that anatomical markers of RGCs may underestimate the extent of RGC dysfunction.

Simulation of Buffered Advection Diffusion of Calcium in a Hepatocyte Cell

The calcium signaling is the basic and vital component of cell communication in almost all types of human and animal cells. All the vital functions of parenchymal cell of liver known as hepatocyte cell are regulated by this calcium signaling. The calcium concentration at specific levels are responsible for each of the various functions of the cell. The deeper understanding of the mechanisms and the factors affecting the calcium dynamics in a hepatocyte cell is vital for various clinical applications related to diseases of the liver. In this paper, mathematical model is proposed to study intracellular calcium dynamics in hepatocyte cell by incorporating the processes like diffusion, advection, buffering etc. The reaction advection diffusion equation has been employed for a two dimensional unsteady state case, to form an initial and boundary value problem. The initial and boundary conditions are formulated based on the physical conditions of cell. Finite volume method and Crank Nicolson scheme have been employed along spatial and temporal dimension respectively to obtain numerical solution. The impact of endogenous and exogenous buffers, advection and diffusion on calcium dynamics in hepatocyte cell has been studied with the help of numerical results. The rise and fall in spatio-temporal calcium concentration in hepatocyte cell in response to specific conditions of advection, diffusion and buffer concentrations is observed. These variations in spatio-temporal calcium concentrations are regulated in narrow range due to fine coordination among these processes of cell under normal environmental and physiological conditions. The proposed model gives better understanding of interrelationship and interdependence of these physical processes for fine coordination among them to maintain structure and functions of cell.

Simultaneous AFM Investigation of the Single Cardiomyocyte Electro-Chemo-Mechanics During Excitation-Contraction Coupling.

The cardiac excitation-contraction coupling is the cellular process through which the heart absolves its blood pumping function, and it is directly affected when cardiac pathologies occur. Cardiomyocytes are the functional units in which this complex biomolecular process takes place: they can be represented as a two-stage electro-chemo and chemo-mechanical transducer, along which each stage can be probed and monitored via appropriate micro/nanotechnology-based tools. Atomic force microscopy (AFM), with its unique nanoresolved force sensitivity and versatile modes of extracting sample properties, can represent a key instrument to study time-dependent heart mechanics and topography at the single cell level. In this work, we show how the integrative possibilities of AFM allowed us to implement an in vitro system which can monitor cardiac electrophysiology, intracellular calcium dynamics, and single cell mechanics. We believe this single cell-sensitive and integrated system will unlock improved, fast, and reliable cardiac in vitro tests in the future.

Analysis of heterogeneous cardiac pacemaker tissue models and traveling wave dynamics

The sinoatrial-node (SAN) is a complex heterogeneous tissue that generates a stable rhythm in healthy hearts, yet a general mechanistic explanation for when and how this tissue remains stable is lacking. Although computational and theoretical analyses could elucidate these phenomena, such methods have rarely been used in realistic (large-dimensional) gap-junction coupled heterogeneous pacemaker tissue models. In this study, we adapt a recent model of pacemaker cells (Severi et al., 2012), incorporating biophysical representations of ion channel and intracellular calcium dynamics, to capture physiological features of a heterogeneous population of pacemaker cells, in particular “center” and “peripheral” cells with distinct intrinsic frequencies and action potential morphology. Large-scale simulations of the SAN tissue, represented by a heterogeneous tissue structure of pacemaker cells, exhibit a rich repertoire of behaviors, including complete synchrony, traveling waves of activity originating from periphery to center, and transient traveling waves originating from the center. We use phase reduction methods that do not require fully simulating the large-scale model to capture these observations. Moreover, the phase reduced models accurately predict key properties of the tissue electrical dynamics, including wave frequencies when synchronization occurs, and wave propagation direction in a variety of tissue models. With the reduced phase models, we analyze the relationship between cell distributions and coupling strengths and the resulting transient dynamics. Further, the reduced phase model predicts parameter regimes of irregular electrical dynamics. Thus, we demonstrate that phase reduced oscillator models applied to realistic pacemaker tissue is a useful tool for investigating the spatial-temporal dynamics of cardiac pacemaker activity.

A microfluidic device with spatiotemporal wall shear stress and ATP signals to investigate the intracellular calcium dynamics in vascular endothelial cells.

Intracellular calcium dynamics plays an important role in the regulation of vascular endothelial cellular functions. In order to probe the intracellular calcium dynamic response under synergistic effect of wall shear stress (WSS) and adenosine triphosphate (ATP) signals, a novel microfluidic device, which provides the adherent vascular endothelial cells (VECs) on the bottom of microchannel with WSS signal alone, ATP signal alone, and different combinations of WSS and ATP signals, is proposed based upon the principles of fluid mechanics and mass transfer. The spatiotemporal profiles of extracellular ATP signals from numerical simulation and experiment studies validate the implementation of our design. The intracellular calcium dynamics of VECs in response to either WSS signal or ATP signal alone, and different combinations of WSS and ATP signals have been investigated. It is found that the synergistic effect of the WSS and ATP signals plays a more significant role in the signal transduction of VECs rather than that from either WSS signal or ATP signal alone. In particular, under the combined stimuli of WSS and ATP signals with different amplitudes and frequencies, the amplitudes and frequencies of the intracellular Ca2+ dynamic signals are observed to be closely related to the amplitudes and frequencies of WSS or ATP signals.

Photocatalytic Activity of Polymer Nanoparticles Modulates Intracellular Calcium Dynamics and Reactive Oxygen Species in HEK-293 Cells.

Optical modulation of living cells activity by light-absorbing exogenous materials is gaining increasing interest, due to the possibility both to achieve high spatial and temporal resolution with a minimally invasive and reversible technique and to avoid the need of viral transfection with light-sensitive proteins. In this context, conjugated polymers represent ideal candidates for photo-transduction, due to their excellent optoelectronic and biocompatibility properties. In this work, we demonstrate that organic polymer nanoparticles, based on poly(3-hexylthiophene) conjugated polymer, establish a functional interaction with an in vitro cell model (Human Embryonic Kidney cells, HEK-293). They display photocatalytic activity in aqueous environment and, once internalized within the cell cytosol, efficiently generate reactive oxygen species (ROS) upon visible light excitation, without affecting cell viability. Interestingly, light-activated ROS generation deterministically triggers modulation of intracellular calcium ion flux, successfully controlled at the single cell level. In perspective, the capability of polymer NPs to produce ROS and to modulate Ca2+ dynamics by illumination on-demand, at non-toxic levels, may open the path to the study of biological processes with a gene-less approach and unprecedented spatio-temporal resolution, as well as to the development of new biotechnology tools for cell optical modulation.

The achievement of boar sperm invitro capacitation is related to an increase of disrupted disulphide bonds and intracellular reactive oxygen species levels.

The aim of this work was to determine the relationship of intracellular reactive oxygen species (ROS) and the disulphide bonds established between sperm proteins with the achievement of capacitation in boar spermatozoa. With this purpose, spermatozoa were incubated in a specifically designed invitro capacitation medium (CM) in the presence or absence of reduced glutathione (GSH). Incubation of boar spermatozoa in CM for 4h significantly (p<0.05) increased free cysteine residues, which is a marker of disrupted disulphide bonds, and also intracellular ROS levels. The addition of GSH to the medium prevented most capacitation-like changes in sperm motility, membrane lipid disorder, mitochondrial membrane potential, intracellular calcium levels and localization of tyrosine-phosphorylated proteins (pTyr), but not in tyrosine phosphorylation of P32. These effects were accompanied by the inhibition of the ability of sperm cells to trigger the acrosome exocytosis in response to progesterone. When GSH was added together with progesterone after 4h of incubation, acrosome exocytosis was not altered, but the subsequent decrease in intracellular calcium observed in controls cells was inhibited. Furthermore, co-incubation of oocytes with spermatozoa previously incubated in CM in the presence of GSH for 4h significantly (p<0.05) increased the number of spermatozoa attached to the oocyte surface but decreased normal fertilization rates. Our results suggest that boar sperm capacitation is related to an increase in disrupted disulphide bonds and intracellular ROS levels and that both events are related to the regulation of hyperactivated motility, intracellular calcium dynamics, sperm binding ability to the oocyte and achievement of proper nuclear decondensation upon oocyte penetration.

A 3D diffusional-compartmental model of the calcium dynamics in cytosol, sarcoplasmic reticulum and mitochondria of murine skeletal muscle fibers.

Variations of free calcium concentration ([Ca2+]) are powerful intracellular signals, controlling contraction as well as metabolism in muscle cells. To fully understand the role of calcium redistribution upon excitation and contraction in skeletal muscle cells, the local [Ca2+] in different compartments needs to be taken into consideration. Fluorescent probes allow the determination of [Ca2+] in the cytosol where myofibrils are embedded, the lumen of the sarcoplasmic reticulum (SR) and the mitochondrial matrix. Previously, models have been developed describing intracellular calcium handling in skeletal and cardiac muscle cells. However, a comprehensive model describing the kinetics of the changes in free calcium concentration in these three compartments is lacking. We designed a new 3D compartmental model of the half sarcomere with radial symmetry, which accounts for diffusion of Ca2+ into the three compartments and simulates its dynamics at rest and at various rates of stimulation in mice skeletal muscle fibers. This model satisfactorily reproduces both the amplitude and time course of the variations of [Ca2+] in the three compartments in mouse fast fibers. As an illustration of the applicability of the model, we investigated the effects of Calsequestrin (CSQ) ablation. CSQ is the main Ca2+ buffer in the SR, localized in close proximity of its calcium release sites and near to the mitochondria. CSQ knock-out mice muscles still preserve a near-normal contractile behavior, but it is unclear whether this is caused by additional SR calcium buffering or a significant contribution of calcium entry from extracellular space, via stored-operated calcium entry (SOCE). The model enabled quantitative assessment of these two scenarios by comparison to measurements of local calcium in the cytosol, the SR and the mitochondria. In conclusion, the model represents a useful tool to investigate the impact of protein ablation and of pharmacological interventions on intracellular calcium dynamics in mice skeletal muscle.

A low‐power digital IC emulating intracellular calcium dynamics

SummaryLow power/area cytomorphic chips may be interfaced and ultimately implanted in the human body for cell‐sensing and cell‐control applications of the future. In such electronic platforms, it is crucial to accurately mimic the biological timescales and operate in real time. This paper proposes a methodology where slow nonlinear dynamical systems describing the behaviour of naturally encountered biological systems can be efficiently realised in hardware. To this end, as a case study, a low power and efficient digital Application‐Specific Integrated Circuit capable of emulating slow intracellular calcium dynamics with timescales reaching to seconds has been fabricated in the commercially available AMS 0.35 μm technology and compared with its analog counterpart. The fabricated chip occupies an area of 1.5 mm2 (excluding the area of the pads) and consumes 18.93 nW for each calcium unit from a power supply of 3.3 V. The presented cytomimetic topology follows closely the behaviour of its biological counterpart, exhibiting similar time‐domain calcium ions dynamics. Results show that the implemented design has the potential to speed up large–scale simulations of slow intracellular dynamics by sharing cellular units in real time.

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

A computational model of excitation and contraction in uterine myocytes from the pregnant rat

Aberrant uterine myometrial activities in humans are major health issues. However, the cellular and tissue mechanism(s) that maintain the uterine myometrium at rest during gestation, and that initiate and maintain long-lasting uterine contractions during delivery are incompletely understood. In this study we construct a computational model for describing the electrical activity (simple and complex action potentials), intracellular calcium dynamics and mechanical contractions of isolated uterine myocytes from the pregnant rat. The model reproduces variant types of action potentials – from spikes with a smooth plateau, to spikes with an oscillatory plateau, to bursts of spikes – that are seen during late gestation under different physiological conditions. The effects of the hormones oestradiol (via reductions in calcium and potassium selective channel conductance), oxytocin (via an increase in intracellular calcium release) and the tocolytic nifedipine (via a block of L-type calcium channels currents) on action potentials and contractions are also reproduced, which quantitatively match to experimental data. All of these results validated the cell model development. In conclusion, the developed model provides a computational platform for further investigations of the ionic mechanism underlying the genesis and control of electrical and mechanical activities in the rat uterine myocytes.

Loss of CaMKI Function Disrupts Salt Aversive Learning in C. elegans.

The ability to adapt behavior to environmental fluctuations is critical for survival of organisms ranging from invertebrates to mammals. Caenorhabditis elegans can learn to avoid sodium chloride when it is paired with starvation. This behavior may help animals avoid areas without food. Although some genes have been implicated in this salt-aversive learning behavior, critical genetic components, and the neural circuit in which they act, remain elusive. Here, we show that the sole worm ortholog of mammalian CaMKI/IV, CMK-1, is essential for salt-aversive learning behavior in C. elegans hermaphrodites. We find that CMK-1 acts in the primary salt-sensing ASE neurons to regulate this behavior. By characterizing the intracellular calcium dynamics in ASE neurons using microfluidics, we find that loss of cmk-1 has subtle effects on sensory-evoked calcium responses in ASE axons and their modulation by salt conditioning. Our study implicates the expression of the conserved CaMKI/CMK-1 in chemosensory neurons as a regulator of behavioral plasticity to environmental salt in C. elegansSIGNIFICANCE STATEMENT Like other animals, the nematode Caenorhabditis elegans depends on salt for survival and navigates toward high concentrations of this essential mineral. In addition to its role as an essential nutrient, salt also causes osmotic stress at high concentrations. A growing body of evidence indicates that C. elegans balances the requirement for salt with the danger it presents through a process called salt-aversive learning. We show that this behavior depends on expression of a calcium/calmodulin-dependent kinase, CMK-1, in the ASE salt-sensing neurons. Our study identifies CMK-1 and salt-sensitive chemosensory neurons as key factors in this form of behavioral plasticity.

3-D Human Sperm Flagellum Tracing in Low SNR Fluorescence Images.

Tracing tubular structures from biomedical images is important for a wide range of applications. Particularly, the spermatozoon is an essential cell whose flagella have a tubular form. Its main function is to fertilize the egg, and the flagellum is fundamental to achieve this task which depends importantly on the dynamics of intracellular calcium ([Ca2+]i). Measuring [Ca2+]i along the flagellum in 3-D is not a simple matter since it requires: 1) sophisticated fluorescence imaging techniques dealing with low intensity and signal to noise ratio (SNR) and 2) tracing the flagellum's centerline. Most of the algorithms proposed to trace tubular structures have been developed for multi-branch structures not being adequate for single tubular structures with low SNR. Taking into account the prior knowledge that the flagellum is constituted by a single tubular structure, we propose an automatic method to trace and track multiple single tubular structures from 3-D images. First, an algorithm based on one-class classification allows enhancement of the flagellum. This enhanced 3-D image permits guiding an iterative centerline algorithm toward the flagellum's centerline. Each sperm is assigned an ID to keep track of it in 3-D . Our algorithm was quantitatively evaluated using a ground truth 564 semi-manual traces (six 3-D image stacks) comparing them to those obtained from state-of-the-art tubular structure centerline extraction algorithms. The qualitative and quantitative results show that our algorithm is extracting similar traces as compared with ground truth, and it is more robust and accurate to trace the flagellum's centerline than multi-branch algorithms.

Rational design of silicon structures for optically controlled multiscale biointerfaces.

Silicon-based materials have been widely used. However, remotely controlled and interconnect-free silicon configurations have been rarely explored, because of limited fundamental understanding of the complex physicochemical processes that occur at interfaces between silicon and biological materials. Here, we describe rational design principles, guided by biology, for establishing intracellular, intercellular and extracellular silicon-based interfaces, where the silicon and the biological targets have matched properties. We focused on light-induced processes at these interfaces, and developed a set of matrices to quantify and differentiate the capacitive, Faradaic and thermal outputs from about 30 different silicon materials in saline. We show that these interfaces are useful for the light-controlled non-genetic modulation of intracellular calcium dynamics, of cytoskeletal structures and transport, of cellular excitability, of neurotransmitter release from brain slices, and of brain activity in vivo.

Optical Probes for Neurobiological Sensing and Imaging.

Fluorescent nanosensors and molecular probes are next-generation tools for imaging chemical signaling inside and between cells. Electrophysiology has long been considered the gold standard in elucidating neural dynamics with high temporal resolution and precision, particularly on the single-cell level. However, electrode-based techniques face challenges in illuminating the specific chemicals involved in neural cell activation with adequate spatial information. Measuring chemical dynamics is of fundamental importance to better understand synergistic interactions between neurons as well as interactions between neurons and non-neuronal cells. Over the past decade, significant technological advances in optical probes and imaging methods have enabled entirely new possibilities for studying neural cells and circuits at the chemical level. These optical imaging modalities have shown promise for combining chemical, temporal, and spatial information. This potential makes them ideal candidates to unravel the complex neural interactions at multiple scales in the brain, which could be complemented by traditional electrophysiological methods to obtain a full spatiotemporal picture of neurochemical dynamics. Despite the potential, only a handful of probe candidates have been utilized to provide detailed chemical information in the brain. To date, most live imaging and chemical mapping studies rely on fluorescent molecular indicators to report intracellular calcium (Ca2+) dynamics, which correlates with neuronal activity. Methodological advances for monitoring a full array of chemicals in the brain with improved spatial, temporal, and chemical resolution will thus enable mapping of neurochemical circuits with finer precision. On the basis of numerous studies in this exciting field, we review the current efforts to develop and apply a palette of optical probes and nanosensors for chemical sensing in the brain. There is a strong impetus to further develop technologies capable of probing entire neurobiological units with high spatiotemporal resolution. Thus, we introduce selected applications for ion and neurotransmitter detection to investigate both neurons and non-neuronal brain cells. We focus on families of optical probes because of their ability to sense a wide array of molecules and convey spatial information with minimal damage to tissue. We start with a discussion of currently available molecular probes, highlight recent advances in genetically modified fluorescent probes for ions and small molecules, and end with the latest research in nanosensors for biological imaging. Customizable, nanoscale optical sensors that accurately and dynamically monitor the local environment with high spatiotemporal resolution could lead to not only new insights into the function of all cell types but also a broader understanding of how diverse neural signaling systems act in conjunction with neighboring cells in a spatially relevant manner.