Ion-sensitive Probes Research Articles

Inorganic ion-sensitive imaging probes for biomedical applications.

Inorganic ions are indispensable substances in living systems, and are widely involved in many essential biological processes. Increasing evidence has shown that the disruption of ion homeostasis is closely related to health problems; thus, the in situ evaluation of ion levels and monitoring their dynamic changes in the living body are critical for precise diagnosis and therapy of diseases. Currently, along with the development of advanced imaging probes, optical imaging and magnetic resonance imaging (MRI) are becoming two major imaging approaches for the identification of ion dynamics. In this review, the design and fabrication of ion-sensitive fluorescent/MRI probes are introduced from the perspective of imaging principles. Furthermore, the recent advances in dynamic imaging of ion levels in living organisms, as well as understanding of ion dyshomeostasis related progression and early diagnosis of diseases, are summarized. Finally, the future perspectives of state-of-the-art ion-sensitive probes for biomedical applications are briefly discussed.

The molecular mechanism of synaptic activity-induced astrocytic volume transient.

Neuronal activity causes astrocytic volume change via K+ uptake through TREK-1 containing two-pore domain potassium channels. The volume transient is terminated by Cl- efflux through the Ca2+ -activated anion channel BEST1. The source of the Ca2+ required to open BEST1 appears to be the stretch-activated TRPA1 channel. Intense neuronal activity is synaptically coupled with a physical change in astrocytes via volume transients. The brain volume changes dynamically and transiently upon intense neuronal activity through a tight regulation of ion concentrations and water movement across the plasma membrane of astrocytes. We have recently demonstrated that an intense neuronal activity and subsequent astrocytic AQP4-dependent volume transient are critical for synaptic plasticity and memory. We have also pharmacologically demonstrated a functional coupling between synaptic activity and the astrocytic volume transient. However, the precise molecular mechanisms of how intense neuronal activity and the astrocytic volume transient are coupled remain unclear. Here we utilized an intrinsic optical signal imaging technique combined with fluorescence imaging using ion sensitive dyes and molecular probes and electrophysiology to investigate the detailed molecular mechanisms in genetically modified mice. We report that a brief synaptic activity induced by a train stimulation (20Hz, 1s) causes a prolonged astrocytic volume transient (80s) via K+ uptake through TREK-1 containing two-pore domain potassium (K2P) channels, but not Kir4.1 or NKCC1. This volume change is terminated by Cl- efflux through the Ca2+ -activated anion channel BEST1, but not the volume-regulated anion channel TTYH. The source of the Ca2+ required to open BEST1 appears to be the stretch-activated TRPA1 channel in astrocytes, but not IP3 R2. In summary, our study identifies several important astrocytic ion channels (AQP4, TREK-1, BEST1, TRPA1) as the key molecules leading to the neuronal activity-dependent volume transient in astrocytes. Our findings reveal new molecular and cellular mechanisms for the synaptic coupling of intense neuronal activity with a physical change in astrocytes via volume transients.

Evaluation of Electron Temperature and Density Using Ion Sensitive Probes on Open Field Plasma in the End-Region of GAMMA 10/PDX

Evaluation of Electron Temperature and Density Using Ion Sensitive Probes on Open Field Plasma in the End-Region of GAMMA 10/PDX

Non-Canonical Control of Neuronal Energy Status by the Na+ Pump

Neurons have limited intracellular energy stores but experience acute and unpredictable increases in energy demand. To better understand how these cells repeatedly transit from a resting to active state without undergoing metabolic stress, we monitored their early metabolic response to neurotransmission using ion-sensitive probes and FRET sensors invitro and invivo. A short theta burst triggered immediate Na+ entry, followed by a delayed stimulation of the Na+/K+ ATPase pump. Unexpectedly, cytosolic ATP and ADP levels were unperturbed across a wide range of physiological workloads, revealing strict flux coupling between the Na+ pump and mitochondria. Metabolic flux measurements revealed a "priming" phase of mitochondrial energization by pyruvate, whereas glucose consumption coincided with delayed Na+ pump stimulation. Experiments revealed that the Na+ pump plays a permissive role for mitochondrial ATP production and glycolysis. We conclude that neuronal energy homeostasis is not mediated by adenine nucleotides or by Ca2+, but by a mechanism commanded by the Na+ pump.

Calibration of intracellular ion probes

The development of ion‐sensitive fluorescent probes enables the measurement of intracellular concentrations of ions (such as H+, K+, Na+, Ca2+, etc.) in a non‐invasive manner. One major complication with their use arises from their unpredictable behavior inside the cell, which makes it necessary to calibrate them in situ. Calibration implies creating known intracellular ion concentrations Ci (for example, making them equal to extracellular concentrations Ce) and relating them to the measured fluorescent signal. The question is how to ensure Ci = Ce?Despite the long history of use of intracellular ion probes, the answer to this question is hard to find in the literature. In fact, many of the published calibration protocols clearly fail to achieve the desired goal. We therefore undertook a theoretical and experimental study of this question using K+ as an example. The general recipe for ion equilibration requires two conditions: (1) cell membrane potential should be destroyed; (2) membrane permeability for both K+ and Na+ should be significantly increased. Our results indicate that a combination of gramicidin, valinomycin, nigericin and ouabain is most suitable for achieving equalization of K+ across the membrane. Calibration curves obtained with suboptimal choice of ionophores leads to erroneous potassium measurements.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bunker probe: A plasma potential probe almost insensitive to its orientation with the magnetic field.

Due to their ability to suppress a large part of the electron current and thus measuring directly the plasma potential, ion sensitive probes have begun to be widely tested and used in fusion devices. For these probes to work, almost perfect alignment with the total magnetic field is necessary. This condition cannot always be fulfilled due to the curvature of magnetic fields, complex magnetic structure, or magnetic field reconnection. In this perspective, we have developed a plasma potential probe (named Bunker probe) based on the principle of the ion sensitive probe but almost insensitive to its orientation with the total magnetic field. Therefore it can be used to measure the plasma potential inside fusion devices, especially in regions with complex magnetic field topology. Experimental results are presented and compared with Ball-Pen probe measurements taken under identical conditions. We have observed that the floating potential of the Bunker probe is indeed little affected by its orientation with the magnetic field for angles ranging from 90° to 30°, in contrast to the Ball-Pen probe whose floating potential decreases towards that of a Langmuir probe if not properly aligned with the magnetic field.

Space-charge limits of ion sensitive probes

Ion sensitive probes (ISPs) are used to measure ion temperature and plasma potential in magnetized plasmas. Their operation relies on the difference in electron and ion Larmor radii to preferentially collect the ion species on a recessed electrode. Because of their simple two-electrode construction and optimal geometry for heat flux handling they are an attractive probe to use in the high heat flux boundary of magnetic confinement fusion experiments. However, the integrity of its measurements is rarely, if ever, checked under such conditions. Recent measurements with an ISP in the Alcator C-Mod tokamak have shown that its ion current is space-charge limited and thus its current–voltage (I–V) response does not contain information on the ion temperature. We numerically solve a 1D Vlasov–Poisson model of ion collection to determine how much bias is needed to overcome space-charge effects and regain the classic I–V characteristic with an exponential decay. Prompted by the observations of space charge in C-Mod, we have performed a survey of ISP measurements reported in the literature. Evidence of space-charge limited current collection is found on many probes, with few authors noting its presence. Some probes are able to apparently exceed the classic 1D space-charge limit because electrons can E × B drift into the probe volume, partially reducing the net ion charge; it is argued that this does not, however, change the basic problem that space charge compromises the measurement of ion temperature. Guidance is given for design of ISPs to minimize the effects of space charge.

Edge and divertor plasma measurements with ion sensitive and Mach probes in LHD

Abstract Spatial profiles of plasma flow and Mach number in the stochastic magnetic boundary layer as well as ion temperature ( T i ) and electron temperature ( T e ) in the divertor region in Large Helical Device (LHD) have been studied by a movable multiple functions probe, which consists of Mach probes and an ion sensitive probe. The results of ion saturation current measurements indicated plasma flow direction is alternated in the stochastic magnetic boundary. Mach number profiles for different plasma densities have been evaluated experimentally which compared with 3-D transport code. T i and T e in the divertor region measured by the ion sensitive probe decreased with increasing line-averaged density. Although T i was higher than T e in the low density plasma, both temperatures became almost the same at higher density.

Multiplexed Sensing of Ions with Barcoded Polyelectrolyte Capsules

Multiplexed detection of analytes is a challenge for numerous medical and biochemical applications. Many fluorescent particulate devices are being developed as ratiometric optical sensors to measure the concentration of intracellular analytes. The response of these sensors is based on changes of the emission intensity of analyte-sensitive probes, entrapped into the carrier system, which depends on the concentration of a specific analyte. However, there are a series of technical limits that prevent their use for quantitative detection of several analytes in parallel (e.g., emission crosstalk between different sensor molecules). Here we demonstrate that double-wall barcoded sensor capsules can be used for multiplexed analysis of proton, sodium, and potassium ions. The sensor detection methodology is based on porous microcapsules which carry ion-sensitive probes in their inner cavity for ion detection and a unique QD barcode in their outermost wall as tag for identification of individual sensors. The engineering of QD barcodes to capsules walls represents a promising strategy for optical multianalyte determination.

Measurement of Ion Parameters by Ion Sensitive Probe in ECR Plasma

Ion parameters in electron cyclotron resonance (ECR) microwave plasma were measured by ion sensitive probe and were compared with the electron parameters obtained by double Langmuir probe. The effects of gas pressure and microwave power on the ion temperature and density were analyzed. The spatial distribution of the ion parameters was also investigated by the ion sensitive probes with a tunable radial depth installed on different probe windows along the chamber axis. Results showed that the ion density measured by the ion sensitive probe was in good agreement with the electron density measured by the double Langmuir probe. The influence of gas pressure on the ion parameters was stronger than that of microwave power. With the increase in working pressure, the ion temperature decreased monotonously with a decreasing rate larger than that at higher pressure. The ion density first increased to a peak (42.3×1010 cm-3) at 1 Pa and then decreased. The ion temperature and density increased little with the increase in the microwave power from 400 W to 800 W. The plasma far away from the resonant point is found to be radially uniform.

Real-Time Imaging of Leaf Apoplastic pH Dynamics in Response to NaCl Stress.

Knowledge concerning apoplastic ion concentrations is important for the understanding of many processes in plant physiology. Ion-sensitive fluorescent probes in combination with quantitative imaging techniques offer opportunities to localize, visualize, and quantify apoplastic ion dynamics in situ. The application of this technique to the leaf apoplast is complicated because of problems associated with dye loading. We demonstrate a more sophisticated dye loading procedure that enables the mapping of spatial apoplastic ion gradients over a period of 3 h. The new technique has been used for the real-time monitoring of pH dynamics within the leaf apoplast in response to NaCl stress encountered by the roots.

Transport of electrons in the tunnel of an ion sensitive probe

Ion sensitive probes serve to measure the ion temperature in magnetized plasma. Such a probe typically consists of a collector submerged inside a hollow tube, which is oriented perpendicularly to the magnetic field. The principle of the probe is based on geometrical shielding of the ion collector from plasma electrons. According to the basic theory, when the collector is retracted in the tube, electrons with their small Larmor radii should not be able to reach it and the collector becomes sensitive to ions. However, experimental results show that the electron shielding is in general inefficient, it only works in the case when the potential of the collector is the same as the potential of the inside surface of the tube.This problem is investigated using a full 3D particle-in-cell Cartesian code with a fast multigrid Poisson solver. We simulate the plasma behaviour in the vicinity of a model of the ion sensitive probe. A positive potential structure is formed at the entrance of the tube due to the space charge of ions that gyrate inside. This structure produces E × B drifts, which push electrons into the shielded space. A stream of electrons hitting the collector is observed for various potentials of the collector. Simulations revealed that electrons can penetrate inside the geometrical shadow in all studied cases; however, they do not reach the collector when the potential of the collector is equal to the potential of the tube.

Ion Temperature Measurement Using Ion-Sensitive Probe

The ion current of ion-sensitive probes is investigated on the basis of a numerical analysis of ion orbits. Each ion-sensitive probe investigated in this study consists of a central electrode and a guard electrode, which are biased to an identical potential, V, against ambient plasma. By fitting an expected function, given by I=I0exp [-c(eV/kBTi)], to numerically obtained V–I characteristics of each ion-sensitive probe, we obtain a calibration coefficient, c, for measurement of ion temperature, Ti, and consider the validity of the fitting. Since the calibration coefficient depends on the radius and height of the guard electrode, the layout of the ion-sensitive probe for a reliable measurement of ion temperature is discussed.

PIC Simulation of the Motion of Plasma around Ion Sensitive Probes

The current-voltage characteristics, the structure of electric potential around an ion sensitive probe and the particle flux on the ion collector have been simulated by the two dimensional particle-in-cell code (Berkeley Code). Concerning the separate mechanism of ions and electrons on the probe, the importance of electric potential profile around the electrode was pointed out. It was found that the E × B drift motion of electrons moving along the equipotential surface plays an essential role in the ISP measurement.

PIC Simulation of the Motion of Plasma around Ion Sensitive Probes

The current-voltage characteristics, the structure of electric potential around an ion sensitive probe and the particle flux on the ion collector have been simulated by the two dimensional particle-in-cell code (Berkeley Code). Concerning the separate mechanism of ions and electrons on the probe, the importance of electric potential profile around the electrode was pointed out. It was found that the E x B drift motion of electrons moving along the equipotential surface plays an essential role in the ISP measurement.

Coupling of Simultaneous Fluorescence and Electrophysiology: Historical Survey, Contributions, and Prospects

The coupling of simultaneous fluorescence and eleclrophysiological measurements paved the way for the development of potentiometric and intracellular free ion-sensitive probes, although the basic and initial aim of these combined studies remains to record fast conformational changes during ion channel activation. Recent high-resolution studies of some channels put back on the agenda challenging methods that combine spectroscopy and electrophysiology. Fluorescence is certainly the most versatile and sensitive spectroscopy in this kind of experiment, and we have recently witnessed significant breakthroughs, at the level of whole cells with intact or mutated channels or with planar lipid bilayers doped with channels or their peptide models. After our initial study of membrane dynamics associated with excitability, following transient pyrene excimer signals during action potentials or voltage-clamp of nerve fibers, we tested the feasability of FRAP (fluorescence recovery after photobleaching) experiments on planar lipid bilayers. This technique was later applied to lateral diffusion measurements of channel forming peptides (alamethicin labeled with fluorescein and the voltage-sensitive segment S4 of the Shaker potassium channel labeled with NBD) under applied voltage. We provide independent evidence for voltage-dependent partitioning and transmembrane insertion and propose renewed experimental avenues to reveal movements and conformational changes associated with ion channel gating and opening.

Lithium induced changes in intracellular free magnesium concentration in isolated rat ventricular myocytes.

We have examined the effect of exposing isolated rat ventricular myocytes to lithium while measuring cytosolic free magnesium ([Mg2+]i) and calcium ([Ca2+]i) levels with the fluorescent, ion sensitive probes mag-fura-2 and fura-2. There was a significant rise in [Mg2+]i after a 5 min exposure to a solution in which 50% of the sodium had been replaced by Li+, but not when the sodium had been replaced by bis-dimethylammonium (BDA). However, there were significant increases in [Ca2+]i when either Na+ substitute was used. The possibility that Li+, which enters the cells, interferes with the signal from mag-fura-2 was eliminated as Li+ concentrations up to 10 mM had no effect on the dye's fluorescence signal. A possible explanation for these findings is that Li+ displaces Mg2+ from intracellular binding sites. Having considered the binding constants for Mg2+ and Li+ to ATP, we conclude that Li+ can displace Mg2+ from Mg-ATP, thus causing a rise in [Mg2+]i. This work has implications for other studies where Li+ is used as a Na+ substitute.

Mechanism of Acidification of the trans-Golgi Network (TGN): IN SITU MEASUREMENTS OF pH USING RETRIEVAL OF TGN38 AND FURIN FROM THE CELL SURFACE

Sorting of secretory cargo and retrieval of components of the biosynthetic pathway occur at the trans-Golgi network (TGN). The pH within the TGN is thought to be an important determinant of these functions. However, studies of the magnitude and regulation of the pH of the TGN have been hampered by the lack of appropriate detection methods. This report describes a noninvasive strategy to measure the luminal pH of the TGN in intact cells. We took advantage of endogenous cellular mechanisms for the specific retrieval of TGN resident proteins, such as TGN38 and furin, that transit briefly to the plasma membrane. Cells were transfected with chimeric constructs that contained the internalization and retrieval signals of TGN resident proteins, and a luminal (extracellular) epitope (CD25). Like TGN38 and furin, the chimeras were shown by fluorescence microscopy to accumulate within the TGN. During their transient exposure at the cell surface, the chimeras were labeled with extracellular anti-CD25 antibodies conjugated with a pH-sensitive fluorophore. Subsequent endocytosis and retrograde transport resulted in preferential labeling of the TGN with the pH-sensitive probe. Continuous, quantitative measurements of the pH of the TGN were obtained by ratio fluorescence imaging. The resting pH, calibrated using either ionophores or the "null point" technique, averaged 5.95 in Chinese hamster ovary cells and 5.91 in HeLa cells. The acidification was dissipated upon addition of concanamycin, a selective blocker of vacuolar-type ATPases. The counterion conductance was found to be much greater than the rate of H+ pumping at the steady state, suggesting that the acidification is not limited by an electrogenic potential. Both Cl- and K+ were found to contribute to the overall counterion permeability of the TGN. No evidence was found for the presence of active Na+/H+ or Ca2+/H+ exchangers on the TGN membrane. In conclusion, selective retrieval of recombinant proteins can be exploited to target ion-sensitive fluorescent probes to specific organelles. The technique provides real-time, noninvasive, and quantitative determinations of the pH, allowing the study of pH regulation within the TGN in intact cells. The acidic pH of the TGN reflects active H+ pumping into an organelle with a low intrinsic H+ permeability and a high conductance to monovalent ions.

A Review of Ion Sensitive Probes

The ion sensitive probe to be presented is an electric probe for the measurement of the ion temperature or the ion energy distribution function of a magnetized plasma. At the same time, the electron temperature and the plasma potential can also be measured. The probe was tested experimentally in a large variety of plasmas, namely, Ne = (106 −1014)/cm3, Ti, Te = (0.05 −100) eV and B = (0.1 −0.5) T. It functioned satisfactorily under the conditions where the ordinary Langmuir probes functioned well. The theory of the separate collection of ions will be presented, laying emphasis on the sheath effect, especially in a dense plasma. Experimentally obtained typical characteristics will be shown refering to critical structural parameters of the probe and corresponding plasma parameters. Results of some cross check experiments in temperature and plasma density measurements will be also reviewed.

Summary of the First Workshop on Electrical Probes in Magnetized Plasmas

The First Workshop on Electrical Probes in Magnetized Plasmas was held in Berlin, Germany, on June 13–15, 1994. Some highlights of the 17 talks are presented here. Although at the time probes were considered to be a reliable method to measure electron temperature, data was presented indicating very low values of the sheath power transmission factor, which can perhaps best be explained by systematic errors in the temperature measurement. A valuable framework for discussing the electron saturation current was provided by a model of current collection based on 100% recycling. Unfortunately, experimental results on the current ratio as a function of angle and the path of the return current were presented which could not be explained within the model. More successful was a model to explain the nonsaturation observed in the ion current as the result of growth of the Debye sheath. Two designs of ion sensitive probes were discussed, without coming to a full understanding of their operation. It was demonstrated that probes swept at very high frequencies can show new phenomena, whereas the averaging required for probes swept slower than the frequency of plasma fluctuations can distort the data.