Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC

Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca2+ (Kd approximately 20 microM, 6 microM and 12 microM respectively) and selectivity over Mg2+ (Kd approximately 2 mM for mag-Fura-2, > 10 mM for Fura-2FF and BTC). Among the tested indicators, BTC was limited by a modest dynamic range upon Ca2+ binding, susceptibility to photodamage, and sensitivity to alterations in pH. All three indicators bound other metal ions including Zn2+, Cd2+ and Gd3+. Interestingly, only in the case of BTC were spectral differences apparent between Ca2+ and other metal ions. For example, the presence of Zn2+ increased BTC fluorescence 6-fold at the Ca2+ isosbestic point, suggesting that this dye may be used as a fluorescent Zn2+ indicator. Fura-2FF has high specificity, wide dynamic range, and low pH sensitivity, and is an optimal low-affinity Ca2+ indicator for most imaging applications. BTC may be useful if experimental conditions require visible wavelength excitation or sensitivity to other metal ions including Zn2+.     

Ratiometric and nonratiometric Ca2+ indicators for the assessment of intracellular free Ca2+ in a breast cancer cell line using a fluorescence microplate reader

Transporters of Ca2+ are potential drug targets and Ca2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca2+ using microplate readers most often use Ca2+ indicators which do not exhibit a spectra shift on Ca2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca2+ levels. However, experimental limitations may limit the use of ratiometric dyes in microplate readers capable of screening. In this study, we compared the assessment of intracellular Ca2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca2+ in the MCF-7 breast cancer cell line and that some of the limitations in the use of fura-2 appear to be overcome by the use of glass bottom microplates. The calibrated intracellular Ca2+ levels derived using fura-2 are consistent with those from microscopy and cuvette-based studies. Fura-2 may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading.     

Micromolar and submicromolar Ca2+ spikes regulating distinct cellular functions in pancreatic acinar cells.

Agonists induce Ca2+ spikes, waves and oscillations initiating at a trigger zone in exocrine acinar cells via Ca2+ release from intracellular Ca2+ stores. Using a low affinity ratiometric Ca2+ indicator dye, benzothiazole coumarin (BTC), we found that high concentrations of agonists transiently increased Ca2+ concentrations to the micromolar range (>10 microM) in the trigger zone. Comparison with results obtained with a high affinity Ca2+ indicator dye, fura-2, indicated that fura-2 was in fact saturated with Ca2+ during the agonist-induced Ca2+ spikes in the trigger zone. We further revealed that the micromolar Ca2+ spikes were necessary for inducing exocytosis of zymogen granules investigated using capacitance measurements. In contrast, submicromolar Ca2+ spikes selectively gave rise to sequential activation of luminal and basal ion channels. These results suggest new functional diversity in Ca2+ spikes and a critical role for the micromolar Ca2+ spikes in exocytotic secretion from exocrine acinar cells. Our data also emphasize the value of investigating the Ca2+ signalling using low affinity Ca2+ indicators.     

Methods for studying store-operated calcium entry

Activation of surface membrane receptors coupled to phospholipase C results in the generation of cytoplasmic Ca2+ signals comprised of both intracellular Ca2+ release, and enhanced entry of Ca2+ across the plasma membrane. A primary mechanism for this Ca2+ entry process is attributed to store-operated Ca2+ entry, a process that is activated by depletion of Ca2+ ions from an intracellular store by inositol 1,4,5-trisphosphate. Our understanding of the mechanisms underlying both Ca2+ release and store-operated Ca2+ entry have evolved from experimental approaches that include the use of fluorescent Ca2+ indicators and electrophysiological techniques. Pharmacological manipulation of this Ca2+ signaling process has been somewhat limited; but recent identification of key molecular players, STIM and Orai family proteins, has provided new approaches. Here we describe practical methods involving fluorescent Ca2+ indicators and electrophysiological approaches for dissecting the observed intracellular Ca2+ signal to reveal characteristics of store-operated Ca2+ entry, highlighting the advantages, and limitations, of these approaches.     

Simultaneous detection of intracellular free calcium and zinc using fura-2FF and FluoZin-3

Elevation of intracellular free zinc ([Zn2+]i) probably contributes to cell death in injury paradigms involving calcium deregulation and oxidative stress such as glutamate excitotoxicity. However, it is difficult to monitor both ions simultaneously in live cells. Here we present a new method using fluorescence microscopy and the ion sensitive indicators fura-2FF and FluoZin-3 to monitor both [Ca2+]i and [Zn2+]i in primary cortical neurons. We show that the new single wavelength dye FluoZin-3 responds robustly to small zinc loads, is insensitive to high Ca2+ or Mg2+, and is relatively unaffected by low pH or oxidants. The ratiometric indicator fura-2FF is sensitive to both Ca2+ and Zn2+. However, in conditions analogous to excitotoxic glutamate exposure where [Ca2+]i is high relative to [Zn2+]i, we found that fura-2FF responds mostly to [Ca2+]i but is relatively unaffected by low [Zn2+]i. Moreover, fura-2FF ratio changes caused by high [Ca2+]i or high [Zn2+]i could be distinguished because each ion produces a different spectral response. Finally, dual dye experiments showed that FluoZin-3 and fura-2FF respond robustly to [Zn2+]i and [Ca2+]j, respectively, in the same neurons during intense glutamate exposure. These studies provide a novel method for the simultaneous detection of both calcium and zinc in cells.     

Iminocoumarin-based low affinity fluorescent Ca2+ indicators excited with visible light.

A series of iminocoumarin-based fluorescent Ca2+ indicators were synthesized and the spectral profiles of their free and Ca2+ bound forms were studied. The newly-synthesized compounds incorporate the Ca2+ chelating structure of BAPTA. The chromophore moieties are iminocoumarins substituted at the 3-position with benzothiazolyl, benzoxazolyl and benzimidazolyl groups. These compounds are excited with visible light and their Ca2+ dissociation constants range from 5.4 to 27.5 microM. Fluorescence spectra studies of these probes indicated a clear shift in their excitation wavelength maxima upon Ca2+ binding along with changes in fluorescence intensity that enable the compounds to be used as low Ca2+ affinity, visible excitable probes.     

New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures

A new family of high-affinity buffers and optical indicators for Ca2+ is rationally designed and synthesized. The parent compound is 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a relative of the well-known chelator EGTA [ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] in which methylene links between oxygen and nitrogen are replaced by benzene rings. BAPTA and its derivatives share the high (greater than 10(5)) selectivity for Ca2+ over Mg2+ of EGTA but are very much less affected by pH changes and are faster at taking up and releasing Ca2+. The affinity of the parent compound for Ca2+ (dissociation constant 1.1 x 10(-7) M in 0.1 M KCl) may be strengthened or weakened by electron-releasing or -withdrawing substituents on the aromatic rings. The Ca2+ and Mg2+ affinities may further be altered by replacing the ether oxygens by heterocyclic nitrogen atoms. The compounds described are fluorescent Ca2+ indicators absorbing in the ultraviolet region; the very large spectral shifts observed on binding Ca2+ fit the prediction that complexation should hinder the conjugation of the nitrogen lone-pair electrons with the aromatic rings. Derivatives with quinoline nuclei are notable for their high sensitivity of fluorescent quantum yield to the binding of Ca2+ but not of Mg2+. Preliminary biological tests have so far revealed little or no binding to membranes or toxic effects following intracellular microinjection.     

Quantitative two-photon Ca2+ imaging via fluorescence lifetime analysis

Two-photon microscopy (TPM) revolutionized Ca2+ imaging by allowing recordings in the depth of intact tissue and live organisms. A serious limitation in TPM, however, is the lack of an accurate and straightforward approach for the quantification of Ca2+ signals, an ability that became an invaluable tool in fluorescence microscopy. Here, we present time-correlated fluorescence lifetime imaging (tcFLIM) as a ratiometric method for the quantification of Ca2+ signals in TPM. The fluorescence lifetime of the Ca2+-indicator dye Oregon Green BAPTA-1 (OGB-1) can be recorded using the approximately 80 MHz excitation pulses utilized in TPM. It shows a Ca2+ dependence that can be explained by the Ca2+-affinity, spectral properties and purity of the dye. Pixel-wise lifetime recordings, controlled by a laser-scanning microscope, allowed quantitative Ca2+ imaging in full-frame and linescan mode. Although we focused on the high-affinity Ca2+ indicator OGB-1, our tcFLIM-based quantification is applicable to other Ca2+ dyes and to fluorescence indicators in general.     

A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals

Quantifying the magnitude of Ca2+ signals from changes in the emission of fluorescent indicators relies on assumptions about the indicator behaviour in situ. Factors such as osmolarity, pH, ionic strength and protein environment can affect indicator properties making it advantageous to calibrate indicators within the required cellular or subcellular environment. Selecting Ca2+ indicators appropriate for a particular application depends upon several considerations including Ca2+ binding affinity, dynamic range and ease of loading. These factors are usually best determined empirically. This study describes the in-situ calibration of a number of frequently used fluorescent Ca2+ indicators (Fluo-3, Fluo-4, Calcium Green-1, Calcium Orange, Oregon Green 488 BAPTA-1 and Fura-Red) and their use in reporting low- and high-amplitude Ca2+ signals in HeLa cells. All Ca2+ indicators exhibited lower in-situ Ca2+ binding affinities than suggested by previously published in-vitro determinations. Furthermore, for some of the indicators, there were significant differences in the apparent Ca2+ binding affinities between nuclear and cytoplasmic compartments. Variation between indicators was also found in their dynamic ranges, compartmentalization, leakage and photostability. Overall, Fluo-3 proved to be the generally most applicable Ca2+ indicator, since it displayed a large dynamic range, low compartmentalization and an appropriate apparent Ca2+ binding affinity. However, it was more susceptible to photobleaching than many of the other Ca2+ indicators.     

Evaluation of the Ca2+ Concentration in Purified Nerve Terminals: Relationship Between Ca2+ Homeostasis and Synaptosomal Preparation

The presynaptic Ca2+ concentration ([Ca]i) was evaluated by studying intracellular free Ca2+ with quin-2 and fura-2 in synaptosomal preparations. The synaptosomal preparations were purified with hyperosmotic (sucrose) and isoosmotic (Percoll) density gradient centrifugation. Synaptosomes are most viable in the heavier fractions of the density gradients. These synaptosomal fractions exhibit the lowest [Ca]i, [204 +/- 2 nM for Percoll (C-band) synaptosomes, loaded at 30 degrees C with the acetoxymethyl ester of fura-2 (fura-2-AM)], a high stability during prolonged incubations at 37 degrees C, and a more potent response to membrane depolarization by elevated extracellular [K+]. [Ca]i measurement was critically dependent on dye loading, calibration, type of dye used, synaptosomal preparation, and incubation temperature (30 degrees or 37 degrees C). Loading quin-2 in synaptosomes inserts a considerable buffer component in the synaptosomal [Ca]i regulation, and consequently there is a quin-2 dependency of [Ca]i, independent of endogenous heavy metal ions. Use of fura-2 is preferable in synaptosomes, although above a critical fura-2-AM/protein ratio during loading ester hydrolysis is not complete, giving rise to errors in [Ca]i determination. Ionomycin is a selective tool to detect the presence of partially hydrolyzed esters and saturate indicators in the cytosol with Ca2+ for calibration. Parallel studies on lactate dehydrogenase and fura-2 fluorescence indicate that synaptosomal viability is very sensitive to prolonged incubations at 37 degrees C. This study shows the applicability of measuring steady-state [Ca]i and dynamic [Ca]i changes quantitatively in fura-2-loaded synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amplification of calcium signals at dendritic spines provides a method for CNS quantal analysis

It has been proposed that the small volume of a dendritic spine can amplify Ca2+ signals during synaptic transmission. Accordingly, we have performed calculations to determine whether the activation of N-methyl-D-aspartate (NMDA) type glutamate receptors during synaptic transmission results in significant elevation in intracellular Ca2+ levels, permitting optical detection of synaptic signals within a single spine. Simple calculations suggest that the opening of even a single NMDA receptor would result in the influx of approximately 310 000 Ca2+ ions into the small volume of a spine, producing changes in Ca2+ levels that are readily detectable using high affinity Ca2+ indicators such as fura-2 or fluo-3. Using fluorescent Ca2+ indicators, we have imaged local Ca2+ transients mediated by NMDA receptors in spines and dendritic shafts attributed to spontaneous miniature synaptic activity. Detailed analysis of these quantal events suggests that the current triggering these transients is attributed to the activation of <10 NMDA receptors. The frequency of these miniature synaptic Ca2+ transients is not randomly distributed across synapses, as some synapses can display a >10-fold higher frequency of transients than others. As expected for events mediated by NMDA receptors, miniature synaptic Ca2+ transients were suppressed by extracellular Mg2+ at negative membrane potentials; however, the Mg2+ block could be removed by depolarization.     

Localization of puff sites adjacent to the plasma membrane: functional and spatial characterization of Ca2+ signaling in SH-SY5Y cells utilizing membrane-permeant caged IP3

The Xenopus oocyte has been a favored model system in which to study spatio-temporal mechanisms of intracellular Ca2+ dynamics, in large part because this giant cell facilitates intracellular injections of Ca2+ indicator dyes, buffers and caged compounds. However, the recent commercial availability of membrane-permeant ester forms of caged IP3 (ci-IP3) and EGTA, now allows for facile loading of these compounds into smaller mammalian cells, permitting control of [IP3]i and cytosolic Ca2+ buffering. Here, we establish the human neuroblastoma SH-SY5Y cell line as an advantageous experimental system for imaging Ca2+ signaling, and characterize IP3-mediated Ca2+ signaling mechanisms in these cells. Flash photo-release of increasing amounts of i-IP3 evokes Ca2+ puffs that transition to waves, but intracellular loading of EGTA decouples release sites, allowing discrete puffs to be studied over a wide range of [IP3]. Puff activity persists for minutes following a single photo-release, pointing to a slow rate of i-IP3 turnover in these cells and suggesting that repetitive Ca2+ spikes with periods of 20-30s are not driven by oscillations in [IP3]. Puff amplitudes are independent of [IP3], whereas their frequencies increase with increasing photo-release. Puff sites in SH-SY5Y cells are not preferentially localized near the nucleus, but instead are concentrated close to the plasma membrane where they can be visualized by total internal reflection microscopy, offering the potential for unprecedented spatio-temporal resolution of Ca2+ puff kinetics.     

Mechanism of specific membrane targeting by C2 domains: localized pools of target lipids enhance Ca2+ affinity

The C2 domain is a ubiquitous, conserved protein signaling motif widely found in eukaryotic signaling proteins. Although considerable functional diversity exists, most C2 domains are activated by Ca2+ binding and then dock to a specific cellular membrane. The C2 domains of protein kinase Calpha (PKCalpha) and cytosolic phospholipase A2alpha (cPLA2alpha), for example, are known to dock to different membrane surfaces during an intracellular Ca2+ signal. Ca2+ activation targets the PKCalpha C2 domain to the plasma membrane and the cPLA2alpha C2 domain to the internal membranes, with no detectable spatial overlap. It is crucial to determine how such targeting specificity is achieved at physiological bulk Ca2+ concentrations that during a typical signaling event rarely exceed 1 muM. For the isolated PKCalpha C2 domain in the presence of physiological Ca2+ levels, the target lipids phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PIP2) are together sufficient to recruit the PKCalpha C2 domain to a lipid mixture mimicking the plasma membrane inner leaflet. For the cPLA2alpha C2 domain, the target lipid phosphatidylcholine (PC) appears to be sufficient to drive membrane targeting to an internal membrane mimic at physiological Ca2+ levels, although the results do not rule out a second, unknown target molecule. Stopped-flow kinetic studies provide additional information about the fundamental molecular events that occur during Ca2+-activated membrane docking. In principle, C2 domain-directed intracellular targeting, which requires coincidence detection of multiple signals (Ca2+ and one or more target lipids), can exhibit two different mechanisms: messenger-activated target affinity (MATA) and target-activated messenger affinity (TAMA). The C2 domains studied here both utilize the TAMA mechanism, in which the C2 domain Ca2+ affinity is too low to be activated by physiological Ca2+ signals in most regions of the cell. Only when the C2 domain nears its target membrane, which provides a high local concentration of target lipid, is the effective Ca2+ affinity increased by the coupled binding equilibrium to a level that enables substantial Ca2+ activation and target docking. Overall, the findings emphasize the importance of using physiological ligand concentrations in targeting studies because super-physiological concentrations can drive docking interactions even when an important targeting molecule is missing.     

Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem

Malaria parasites, Plasmodia, spend most of their asexual life cycle within red blood cells, where they proliferate and mature. The erythrocyte cytoplasm has very low [Ca2+] (<100 nM), which is very different from the extracellular environment encountered by most eukaryotic cells. The absence of extracellular Ca2+ is usually incompatible with normal cell functions and survival. In the present work, we have tested the possibility that Plasmodia overcome the limitation posed by the erythrocyte intracellular environment through the maintenance of a high [Ca2+] within the parasitophorous vacuole (PV), the compartment formed during invasion and within which the parasites grow and divide. Thus, Plasmodia were allowed to invade erythrocytes in the presence of Ca2+ indicator dyes. This allowed selective loading of the Ca2+ probes within the PV. The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism. We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV. A transient drop of [Ca2+] in the PV for a period as short as 2 h affects the maturation process of the parasites within the erythrocytes, with a major reduction 48 h later in the percentage of schizonts, the form that re-invades the red blood cells.     

Modeling Ca2+ signaling differentiation during oocyte maturation

Ca2+ is a fundamental intracellular signal that mediates a variety of disparate physiological functions often in the same cell. Ca2+ signals span a wide range of spatial and temporal scales, which endow them with the specificity required to induce defined cellular functions. Furthermore, Ca2+ signaling is highly plastic as it is modulated dynamically during normal physiological development and under pathological conditions. However, the molecular mechanisms underlying Ca2+ signaling differentiation during cellular development remain poorly understood. Oocyte maturation in preparation for fertilization provides an exceptionally well-suited model to elucidate Ca2+ signaling regulation during cellular development. This is because a Ca2+ signal with specialized spatial and temporal dynamics is universally essential for egg activation at fertilization. Here we use mathematical modeling to define the critical determinants of Ca2+ signaling differentiation during oocyte maturation. We show that increasing IP3 receptor (IP3R) affinity replicates both elementary and global Ca2+ dynamics observed experimentally following oocyte maturation. Furthermore, our model reveals that because of the Ca2+ dependency of both SERCA and the IP3R, increased IP3R affinity shifts the system's equilibrium to a new steady state of high cytosolic Ca2+, which is essential for fertilization. Therefore our model provides unique insights into how relatively small alterations of the basic molecular mechanisms of Ca2+ signaling components can lead to dramatic alterations in the spatio-temporal properties of Ca2+ dynamics.     

Calcium signaling as a novel method to optimize the biosynthetic response of chondrocytes to dynamic mechanical loading

Chondrocyte sensitization and desensitization to mechanical stimuli are complex phenomena that have not been fully described. In this study, we investigated the temporal response of chondrocytes to dynamic mechanical loading and whether changes in calcium signaling could be used a predictor of the biosynthetic response. Cell-seeded agarose gels pre-incubated with an intracellular \(\hbox {Ca}^{2+}\) dye (Fluo-4) were subjected to dynamic compressive loading under varying conditions (amplitude and duration). Induced changes in \(\hbox {Ca}^{2+}\) signaling were determined by confocal imaging and matrix biosynthesis by radioisotope incorporation. It was observed that chondrocytes required a minimum amount of stimulation in order to elicit an anabolic response and they quickly became insensitive to the imposed stimulus. The response appeared to be amplitude dependent and could be predicted by measuring resultant changes in \(\hbox {Ca}^{2+}\) signaling. A positive correlation between \(\hbox {Ca}^{2+}\) signaling and matrix synthesis was achieved when changes in \(\hbox {Ca}^{2+}\) signaling was expressed as a relative number of cells experiencing multiple transients. In addition, these changes in \(\hbox {Ca}^{2+}\) signaling were effective at determining optimal recovery period between successive applications of intermittent mechanical loading, in which full mechanosensitivity was achieved when \(\hbox {Ca}^{2+}\) signaling was allowed to return to baseline (control) levels. The use of \(\hbox {Ca}^{2+}\) signaling to predict the effectiveness of a particular mechanical stimulus as well as to determine optimal refractory periods appears to be advantageous over empirical-based approaches. Future work will investigate the process of \(\hbox {Ca}^{2+}\) ion sequestration into intracellular stores to elucidate potential desensitization mechanisms to dynamic mechanical loading.     

Two modes of inhibition of the Ca2+ pump in red cells by Ca2+

Two different and independent modes of inhibition of the Ca2+ pump by Ca2+ can be detected measuring active Ca2+ extrusion from resealed ghosts of human red cells: one requires extracellular and the other requires intracellular Ca2+. Ki for inhibition by extracellular Ca2+ is about 10 mM. Extracellular Mg2+ replaces Ca2+ in inhibiting Ca2+ transport but with an apparent affinity for inhibition about 3-times less than that for Ca2+. Inhibition by external Ca2+ is not affected by Na+ or K+ at both surfaces of the cell membrane, external EGTA, internal Ca2+ or ATP. The apparent affinity for external Ca2+ progressively raises as pH increases. The effects of extracellular Ca2+ and Mg2+ are consistent with the idea that for Ca2+ pumping to proceed, external sites in the pump must be protonated and not occupied by extracellular Ca2+ or Mg2+. Inhibition by intracellular Ca2+ takes place with a Ki of about 1 mM and is independent of external Ca2+. The inhibitory effects of intracellular Ca2+ can be accounted for if Ca2+ and CaATP were competitive inhibitors of the activation of the pump by Mg2+ and MgATP, respectively.     

A new generation of Ca2+ indicators with greatly improved fluorescence properties

A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.     

培养的大鼠海马神经元胞内Ca2＋和 NO 双标记方法研究

以培养 8 ～ 10 天的大鼠海马神经元为对象,选择 Calcium Orange AM 和 DAF-FM diacetate 为 Ca2＋和一氧化氮 (NO) 的荧光指示剂,建立了基于激光扫描共聚焦显微技术的细胞内 Ca2＋和 NO 双标记检测方法 . 此方法对 Ca2＋和 NO 进行分步染色,然后应用激光扫描共聚焦显微镜 (LSCM) 的双轨迹 (Two Track) 模式,通过快速切换激光实现对细胞内 Ca2＋和 NO 的同时检测 . 实验结果显示,两种染料之间无串扰现象;在 N- 甲基 -D- 天冬氨酸 (NMDA) 刺激下,海马神经元胞内 Ca2＋快速升高,随后达到平台期并有波动, NO 则稳定持续升高,这些变化过程与单标记的结果一致;双标记层切序列图像显示细胞内 Ca2＋和 NO 都较集中分布于细胞中部,但在细节上两者的分布存在差异 . 此双标记方法能同时检测培养的海马神经元胞内 Ca2＋和 NO ,为研究神经元胞内 Ca2＋和 NO 的相互调控作用提供了一种新的手段 .     

Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers.

Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators (mag-fura-5, mag-indo-1, and magnesium orange), the change in fluorescence in response to an action potential (delta F) appeared to track the myoplasmic Ca2+ transient (delta[Ca2+]) without delay. With three lower-affinity tetracarboxylate indicators (BTC, calcium-orange-5N, and calcium-green-5N) and one tricarboxylate indicator (magnesium green), delta F responded to delta[Ca2+] with a small delay. Unfortunately, with the tetracarboxylate indicators, other problems were detected that appear to limit their usefulness as reliable Ca2+ indicators. Surprisingly, delta F from mag-fura-red, another tricarboxylate indicator, was biphasic (with 480 nm excitation), a feature that also greatly limits its usefulness. With several of the indicators, estimates were obtained for the myoplasmic value of KD, Ca (the indicator's dissociation constant for Ca2+) and found to be elevated severalfold in comparison with the value measured in a simple salt solution. These and other problems related to the quantitative use of Ca2+ indicators in the intracellular environment are evaluated and discussed.