Origins of Ca2+ Imaging with Fluorescent Indicators.

In 1980, Roger Tsien published a paper, in this journal [Tsien, R. Y. (1980) Biochemistry, 19 (11), 2396], titled "New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures". These new buffers included 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or BAPTA, which is still widely used today. And so, the world was set alight with new ways in which to visualize Ca2+. The ability to watch fluctuations in intracellular Ca2+ revolutionized the life sciences, although the fluorescent indicators used today, particularly in neurobiology, no longer rely exclusively on BAPTA but on genetically encoded fluorescent Ca2+ indicators. In this Perspective, we reflect on the origins of Ca2+ imaging with a special focus on the contributions made by Roger Tsien, from the early concept of selective Ca2+ binding described in Biochemistry to optical Ca2+ indicators based on chemically synthesized fluorophores to genetically encoded fluorescent Ca2+ indicators.

Effect of Physiological Concentrations of Vitamin C on the Inhibitation of Hydroxyl Radical Induced Light Emission from Fe2+-EGTA-H2O2 and Fe3+-EGTA-H2O2 Systems In Vitro.

Ascorbic acid (AA) has antioxidant properties. However, in the presence of Fe2+/Fe3+ ions and H2O2, it may behave as a pro-oxidant by accelerating and enhancing the formation of hydroxyl radicals (•OH). Therefore, in this study we evaluated the effect of AA at concentrations of 1 to 200 µmol/L on •OH-induced light emission (at a pH of 7.4 and temperature of 37 °C) from 92.6 µmol/L Fe2+-185.2 µmol/L EGTA (ethylene glycol-bis (ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid)-2.6 mmol/L H2O2, and 92.6 µmol/L Fe3+-185.2 µmol/L EGTA-2.6 mmol/L H2O2 systems. Dehydroascorbic acid (DHAA) at the same range of concentrations served as the reference compound. Light emission was measured with multitube luminometer (AutoLumat Plus LB 953) for 120 s after automatic injection of H2O2. AA at concentrations of 1 to 50 µmol/L and of 1 to 75 µmol/L completely inhibited light emission from Fe2+-EGTA-H2O2 and Fe3+-EGTA-H2O2, respectively. Concentrations of 100 and 200 µmol/L did not affect chemiluminescence of Fe3+-EGTA-H2O2 but tended to increase light emission from Fe2+-EGTA-H2O2. DHAA at concentrations of 1 to 100 µmol/L had no effect on chemiluminescence of both systems. These results indicate that AA at physiological concentrations exhibits strong antioxidant activity in the presence of chelated iron and H2O2.

Cal-520FF is the Present Optimal Ca2+ Indicator for Ultrafast Ca2+ Imaging and Optical Measurement of Ca2+ Currents.

Ultrafast Ca2+ imaging using low-affinity fluorescent indicators allows the precise measurement of the kinetics of fast Ca2+ currents mediated by voltage-gated Ca2+ channels. Thus far, only a few indicators provided fluorescence transients with sufficient signal-to-noise ratio necessary to achieve this measurement, with Oregon Green BAPTA-5N exhibiting the best performance. Here we evaluated the performance of the low-affinity Ca2+ indicator Cal-520FF to record fast Ca2+ signals and to measure the kinetics of Ca2+ currents. Compared to Oregon Green BAPTA-5N and to Fluo4FF, Cal-520FF offers a superior signal-to-noise-ratio providing the optimal characteristics for this important type of biophysical measurement. This ability is the result of a relatively high fluorescence at zero Ca2+, necessary to detect enough photons at short exposure windows, and a high dynamic range leading to large fluorescence transients associated with short Ca2+ influx periods. We conclude that Cal-520FF is at present the optimal commercial low-affinity Ca2+ indicator for ultrafast Ca2+ imaging applications.

Bringing the Ca2+ sensitivity of myristoylated recoverin into the physiological range.

The prototypical Ca2+-sensor protein recoverin (Rec) is thought to regulate the activity of rhodopsin kinase (GRK1) in photoreceptors by switching from a relaxed (R) disc membrane-bound conformation in the dark to a more compact, cytosol-diffusing tense (T) conformation upon cell illumination. However, the apparent affinity for Ca2+ of its physiologically relevant form (myristoylated recoverin) is almost two orders of magnitude too low to support this mechanism in vivo. In this work, we compared the individual and synergistic roles of the myristic moiety, the GRK1 target and the disc membrane in modulating the calcium sensitivity of Rec. We show that the sole presence of the target or the disc membrane alone are not sufficient to achieve a physiological response to changes in intracellular [Ca2+]. Instead, the simultaneous presence of GRK1 and membrane allows the T to R transition to occur in a physiological range of [Ca2+] with high cooperativity via a conformational selection mechanism that drives the structural transitions of Rec in the presence of multiple ligands. Our conclusions may apply to other sensory transduction systems involving protein complexes and biological membranes.

Useful Caged Compounds for Cell Physiology.

Light has been instrumental in the study of living cells since its use helped in their discovery in the late 17th century. Further, combining chemical technology with light microscopy was an essential part of the Nobel Prize for Physiology in 1906. Such landmark scientific findings involved passive observation of cells. However, over the past 50 years, a "second use" of light has emerged in cell physiology, namely one of rational control. The seminal method for this emerged in late 1970s with the invention of caged compounds. This was the point when "caged compounds" were defined as optical probes in which the active functionality of a physiological signaling molecule was blocked with a photochemical protecting group. Caged compounds are analogous to prodrugs; in both, the activity of the effector is latent. However, caged compounds, unlike prodrugs, use a trigger that confers the power of full temporal and spatial manipulation of the effects of release of its latent biological cargo. Light is distinct because it is bio-orthogonal, passes through living tissue (even into the cell interior), and initiates rapid release of the "caged" biomolecule. Further, because light can be directed to broad areas or focused to small points, caged compounds offer an array of timing scenarios for physiologists to dissect virtually any type of cellular process.The collaborative interaction between chemists and physiologists plays a fundamental role in the development of caged compounds. First, the physiologists must define the problem to be addressed; then, with the help of chemists, decide if a caged compound would be useful. For this, structure-activity relationships of the potential optical probe and receptor must be determined. If rational targets seem feasible, synthetic organic chemistry is used to make the caged compound. The crucial property of prephotolysis bio-inertness relies on physiological or biochemical assays. Second, detailed optical characterization of the caged compound requires the skill of photochemists because the rate and efficiency of uncaging are also crucial properties for a useful caged compound. Often, these studies reveal limitations in the caged compound which has been developed; thus, chemists and physiologists use their abilities for iterative development of even more powerful optical probes. A similar dynamic will be familiar to scientists in the pharmaceutical industry. Therefore, caged compound development provides an excellent training framework for (young) chemists both intellectually and professionally. In this Account, I draw on my long experience in the field of making useful caged compounds for cell physiology by showing how each probe I have developed has been defined by an important physiological problem. Fundamental to this process has been my initial training by the pioneers in aromatic photochemistry, Derek Bryce-Smith and Andrew Gilbert. I discuss making a range of "caged calcium" probes, ones which went on to be the most widely used of all caged compounds. Then, I describe the development of caged neurotransmitters for two-photon uncaging microscopy. Finally, I survey recent work on making new photochemical protecting groups for wavelength orthogonal, two-color, and ultraefficient two-photon uncaging.

Modeling and simulation of spatial-temporal calcium distribution in T lymphocyte cell by using a reaction-diffusion equation.

T lymphocytes are white blood cells that play a central role in cell-mediated immunity. Ca2+ has its major signaling function when it is elevated in the cytosolic compartment. The free cytosolic Ca2+ dynamics plays a very important role in the activation, and fate decision process in the T lymphocytes. Here, we develop a quantitative spatio-temporal Ca2+ dynamic model which includes, the Ca2+ releasing channels ER leak and voltage-gated Ca2+ channel, buffering and re-uptaking mechanism in the T lymphocytes. In this model, the cell is represented as a circular-shaped geometrical domain. This representation introduces modeling flexibility needed for detailed representation of the properties of Ca2+ dynamics in the cell including important parameters. The proposed mathematical model is solved using a finite difference method and the finite element method. Appropriate initial and boundary conditions are incorporated in the model based on biophysical conditions of the problem. Computer simulations in MATLAB R2010a are employed to investigate mathematical models of reaction-diffusion equation. The estimation is based on reaction-diffusion equation associated with biophysical and biochemical reactions taking place in the cell. From our results, it is observed that, the coordinated combination of the incorporated parameters plays a significant role in Ca2+ regulation in T lymphocytes. ER leak and voltage-gated Ca2+ channel provides the necessary Ca2+ to the cell when required for its proper functioning, while on the other side buffers and Na+/Ca2+ exchanger makes balance in the Ca2+ concentration, so as to prevent the cell from death as higher concentration for longer time is harmful for the cell and can cause cell death. These results have been used to study the relationship of Ca2+ concentration with parameters like VGCC, Na+/Ca2+ exchanger, ER leak and buffers. The significance of the study reveals that there is a significant variation in Ca2+ profiles due to the effect of VGCC, Na+/Ca2+ exchanger, ER leak, and buffers. The results give us better insights of coordinated effect of VGCC, Na+/Ca2+ exchanger, ER leak, and buffers on Ca2+ distribution in T lymphocytes. T lymphocytes are the primary host cells to receive the viral infections which transmits the signal then to other cell types. The proper quantity of Ca2+ concentration makes T lymphocytes more active and healthier to fight the infection properly and can protect the immune system from various fatal viral infections. Thus, the application of the study lies in the field of immunology to protect a susceptible from various viral infectious diseases like HIV, HBV, HINI, etc. by strengthening the immune system. The outcomes of the study reveal that the applied finite element method is computationally very strong and effective to analyze differential equations that arise in Ca2+ dynamics.

Mechanism of Ca2+-mediated NOX modulated in ROS metabolism induced by T-2 toxin in potato tuber.

T-2 toxin at low concentrations can induce ROS accumulation and modulate host resistance in plants. NOX plays crucial roles in ROS production and is regulated by Ca2+via direct binding to EF-hand motifs. In this study, the effect of EGTA (Ca2+ chelating agent) on the expression and enzymatic activity of NOX, as well as the activities and corresponding gene expressions involved in ROS metabolism and cell membrane integrity, were investigated in treated slices. Results indicated that EGTA treatment significantly affected gene expression and activity of NOX, and reduced ROS accumulation and cell membrane integrity and the enzymatic activities and gene expression involved in ROS metabolism when exposed to treatment. The addition of exogenous Ca2+ restored the initial relative transcript abundance, ROS accumulation and their activities. Results suggest that Ca2+ affected by EGTA plays a crucial role in NOX activity regulation, ultimately affecting ROS metabolism in slices induced by T-2 toxin.

The regulatory protein 14-3-3ß binds to the IQ motifs of myosin-IC independent of phosphorylation.

Myosin-IC (Myo1c) has been proposed to function in delivery of glucose transporter type 4 (GLUT4)-containing vesicles to the plasma membrane in response to insulin stimulation. Current evidence suggests that, upon insulin stimulation, Myo1c is phosphorylated at Ser701, leading to binding of the signaling protein 14-3-3ß. Biochemical and functional details of the Myo1c-14-3-3ß interaction have yet to be described. Using recombinantly expressed proteins and mass spectrometry-based analyses to monitor Myo1c phosphorylation, along with pulldown, fluorescence binding, and additional biochemical assays, we show here that 14-3-3ß is a dimer and, consistent with previous work, that it binds to Myo1c in the presence of calcium. This interaction was associated with dissociation of calmodulin (CaM) from the IQ motif in Myo1c. Surprisingly, we found that 14-3-3ß binds to Myo1c independent of Ser701 phosphorylation in vitro Additionally, in contrast to previous reports, we did not observe Myo1c Ser701 phosphorylation by Ca2+/CaM-dependent protein kinase II (CaMKII), although CaMKII phosphorylated four other Myo1c sites. The presence of 14-3-3ß had little effect on the actin-activated ATPase or motile activities of Myo1c. Given these results, it is unlikely that 14-3-3ß acts as a cargo adaptor for Myo1c-powered transport; rather, we propose that 14-3-3ß binds Myo1c in the presence of calcium and stabilizes the calmodulin-dissociated, nonmotile myosin.

Elimination of Intracellular Calcium Overload by BAPTA-AM-Loaded Liposomes: A Promising Therapeutic Agent for Acute Liver Failure.

In the past few decades, intracellular calcium overload has been shown to induce cell death through multiple signaling pathways. In this study, we used BAPTA-AM, a well-known membrane-permeable Ca2+ chelator, to prevent cell injury by allaying the intracellular calcium overload. We explored the clinical potentials of BAPTA-AM-loaded liposome (BAL) in the treatment of the acute liver failure (ALF) mouse model, which is characterized by severe hepatic necrosis and apoptosis. We discovered that BAL can significantly inhibit D-GalN-induced LO2 cell damage as it increased cell viability by 60% and downregulated the LPS-stimulated inflammatory response in RAW 264.7 macrophages by reversing the morphological change and modulating TNF-α and NF-κB expressions. Through systemic administration, BAL can rapidly accumulate in damaged liver tissue and exhibit excellent treatment effects on the D-GalN/LPS-induced ALF mouse model, including elevation of the survival rate (from 10 to 80%), recovery of normal liver indexes and liver health indicators, improvement of liver blood microcirculation (increased the blood flow volume by 80% and flow rate by 60%), and blood coagulation. The underlying hepatoprotective effect of BAL is presumably based on the antinecrosis and antiapoptosis abilities attributed to its inhibition on oxidative stress, restriction on TNF-α receptor, and mitochondria-mediated apoptotic pathway by effectively clearing the overloaded intercellular calcium. BAL holds great potential as a new therapeutic strategy for ALF treatment, and its prominent cell rescue ability provides ample opportunities for the treatment of many other diseases that are characterized by rapid and massive cell damage.

Development of Zinc-Specific iCEST MRI as an Imaging Biomarker for Prostate Cancer.

The healthy prostate contains the highest concentration of mobile zinc in the body. As this level decreases dramatically during the initial development of prostate cancer, in vivo detection of prostate zinc content may be applied for diagnosis of prostate cancer. Using 19 F ion chemical exchange saturation transfer magnetic resonance imaging (iCEST MRI) and TF-BAPTA as a fluorinated Zn-binding probe with micromolar sensitivity, we show that iCEST MRI is able to differentiate between normal and malignant prostate cells with a 10-fold difference in contrast following glucose-stimulated zinc secretion in vitro. The iCEST signal decreased in normal prostate cells upon downregulation of the ZIP1 zinc transporter. In vivo, using an orthotopic prostate cancer mouse model and a transgenic adenocarcinoma of the mouse prostate (TRAMP) model, a gradual decrease of >300 % in iCEST contrast following the transition of normal prostate epithelial cells to cancer cells was detected.

Human Bone Paleoproteomics Utilizing the Single-Pot, Solid-Phase-Enhanced Sample Preparation Method to Maximize Detected Proteins and Reduce Humics.

Sample preparation has become an important part of bone proteomics and paleoproteomics and remains one of the major challenges to maximizing the number of proteins characterized from bone extractions. Most paleoproteomic studies have relied on in-solution digestion with the inclusion of filter-aided sample preparation (FASP) as effective methods to detect the proteome. However, neither of these are optimal because few proteins have been detected utilizing only in-solution digestion and the molecular weight cutoff of FASP may miss remaining fragments of proteins in fossil bone. The recently developed single-pot, solid-phase-enhanced sample preparation (SP3) overcomes these issues by not relying on molecular weight while still controlling where the proteins are digested. Here, historical human bones were extracted with either 500 mM tetrasodium EDTA or 400 mM ammonium phosphate dibasic, 200 mM ammonium bicarbonate, 4 M guanidine HCl and digested with the SP3 method. Across all samples, 78 ± 7 (400-200-4) and 79 ± 17 (EDTA) protein accessions were identified, including previously difficult to detect proteins such as osteopontin. SP3 also effectively removed 90% or more of the coextracting humic substances (based on reduced absorbance) from extracted proteins. The utility of SP3 for maximizing the number of protein detections in historical bones is promising for future paleoproteomic studies.

Monitoring glucose, calcium, and magnesium levels in saliva as a non-invasive analysis by sequential injection multi-parametric determination.

The use of saliva for diagnose and surveillance of systemic illnesses, and general health has been arousing great interest worldwide, emerging as a highly desirable goal in healthcare. The collection is non-invasive, stress-free, inexpensive, and simple representing a major asset. Glucose, calcium, and magnesium concentration are three major parameters evaluated in clinical context due to their essential role in a wide range of biochemical reactions, and consequently many health disorders. In this work, a spectrophotometric sequential injection method is described for the fast screening of glucose, calcium, and magnesium in saliva samples. The glucose determination reaction involves the oxidation of the aldehyde functional group present in glucose with simultaneous reduction of 3,5-dinitrosalicylic acid (DNS) to 3-amino, 5-nitrosalicylic acid under alkaline conditions, followed by the development of colour. The determination of both metals is based on their reaction with cresolphtalein complexone (CPC), and the interference of calcium in the magnesium determination minimized by ethylene glycol-bis[ß-aminoethyl ether]-N,N,N',N'-tetraacetic acid (EGTA). The developed multi-parametric method enabled dynamic ranges of 50 - 300 mg/dL for glucose, 0.1 - 2 mg/dL for calcium, and 0.1 - 0.5 mg/dL for magnesium. Determination rates of 28, 60, 52 h-1 were achieved for glucose, calcium, and magnesium, respectively. Less than 300 µL of saliva is required for the multi-parametric determination due to saliva viscosity and inherent necessity of dilution prior to analysis. RSDs lower than 5% were obtained, and the results agreed with those obtained by reference methods, while recovery tests confirmed its accuracy.

Light Emission from the Fe2+-EGTA-H2O2 System: Possible Application for the Determination of Antioxidant Activity of Plant Phenolics.

Oxidative reactions can result in the formation of electronically excited species that undergo radiative decay depending on electronic transition from the excited state to the ground state with subsequent ultra-weak photon emission (UPE). We investigated the UPE from the Fe2+-EGTA (ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid)-H2O2 system with a multitube luminometer (Peltier-cooled photon counter, spectral range 380 to 630 nm). The UPE of 92.6 µmol/L Fe2+-185.2 µmol/L EGTA-2.6 mmol/L H2O2 reached 4319 ± 755 relative light units during 2 min measurement and was about seven times higher (p < 0.001) than the UPE of incomplete systems (Fe2+-H2O2, EGTA-H2O2) and medium alone. Substitution of Fe2+ with Cr2+, Co2+, Mn2+ or Cu2+ as well as of EGTA with EDTA (ethylenediaminetetraacetic acid) or citrate completely abolished UPE. Experiments with ROS scavengers revealed the dependence of UPE on hydroxyl radicals suggesting occurrence of oxidative attack and cleavage of the ether bond in EGTA backbone structure and formation of triplet excited carbonyl groups with subsequent light emission. Plant phenolics (ferulic, chlorogenic and caffec acids) at concentration 87 µmol/L and ascorbate at 0.46 mmol/L inhibited UPE by 90 ± 4%, 90 ± 5%, 97 ± 2% and 92 ± 1%, respectively. Quenching of UPE from Fe2+-EGTA-H2O2 system can be used for evaluation of antioxidant activity of phytochemicals.

Uptake of iron (III)-ethylenediamine-N, N, N', N'-tetraacetic acid complex by phosphatidylcholine lipid film. Part II. Effect of film curvature.

Mixed micelles formed in a ternary-solute aqueous solution of NaOH, iron (III)-ethylenediamine-N, N, N', N'-tetraacetic acid complex (Fe-EDTA) and 1,2-diheptanoyl-sn-glycero-3-phosphatidyl choline (DHPC) were studied and compared with the mixed adsorbed film reported in Part I of this series to clarify the effect of the curvature of molecular assemblies on the interactions between their Fe-EDTA and DHPC constituents. The critical micelle concentrations (CMCs), surface tension at the CMC, and solution pH were measured as functions of the mole fractions of NaOH and DHPC. Rigorous thermodynamic equations were derived, in which the overall proton dissociation equilibria of Fe-EDTA and DHPC were taken into consideration, and applied to experimental data to obtain phase diagrams of micelle formation and the micelle-adsorbed film equilibrium. It was found that when the bulk solution was strongly acidic, Fe-EDTA was incorporated in the micelles. However, the adsorbed film was more Fe-EDTA-enriched than the micelle. These findings imply that a flat cell membrane is more permeable to an iron complex than a cell membrane with positive curvature.

Uptake of iron (III)-ethylenediamine-N,N,N',N'-tetraacetic acid complex by phosphatidylcholine lipid film: Part I. Effect of bulk pH.

We studied a ternary solutes aqueous solution of NaOH, iron (III)-ethylenediamine-N,N,N',N'-tetraacetic acid complex (Fe-edta), and 1,2-diheptanoyl-sn-glycero-3-phosphatidylcholine (DHPC)/air interface system to clarify the interactions between iron complexes and lipids with a phosphatidylcholine head group. The solution surface tension and pH were measured as functions of the total molality of NaOH, Fe-edta and DHPC, and the mole fractions of NaOH and DHPC. Rigorous thermodynamic equations were derived, in which the overall proton dissociation equilibria of Fe-edta and DHPC were taken into consideration, and applied to experimental data to obtain phase diagram of adsorption. It was found that (1) adsorption of Fe-edta at the solution/air interface with a DHPC monolayer was about 50-130 times higher than that without a DHPC monolayer and (2) when the bulk mole fraction of NaOH was high, Fe-edta tended to be expelled from the adsorbed film. The last finding suggests that the ambient pH significantly affects passive transport of the iron complex through a phospholipid-containing membrane into the cell interior.

Diosgenin, an Activator of 1,25D3-MARRS Receptor/ERp57, Attenuates the Effects of TNF-α by Causing ADAM10-Dependent Ectodomain Shedding of TNF Receptor 1.

BACKGROUND/AIMS: We investigated how diosgenin, a steroidal sapogenin, has anti-tumor necrosis factor-α (TNF-α) effects in human aortic endothelial cells (HAECs). METHODS: Tumor necrosis factor receptor 1 (TNFR1) was assessed by Western blot analysis. Intracellular Ca2+ was measured using Fluo-4 AM. Immunofluorescence staining was performed for a disintegrin and metalloprotease 10 (ADAM10). RESULTS: Diosgenin (1 ∼ 100 nM) induced ectodomain shedding of TNFR1 within 30 min and attenuated TNF-α-induced intercellular adhesion molecule-1 (ICAM-1) expression. Upon treatment with diosgenin, extracellular Ca2+ entered into the cells via L-type calcium channels, whereas diosgenin-induced ectodomain shedding of TNFR1 was almost completely inhibited by BAPTA-AM (intracellular Ca2+ chelator), verapamil (L-type calcium channel antagonist) and the absence of extracellular Ca2+. Diosgenin caused translocation of ADAM10 to the cell surface, which was mediated by extracellular Ca2+ influx. Depletion of ADAM10 prevented diosgenin-induced ectodomain shedding of TNFR1 and abolished the inhibitory effect of diosgenin on TNF-α-induced ICAM-1 expression. Diosgenin did not induce extracellular Ca2+ influx and ectodomain shedding of TNFR1 in cells depleted of 1,25D3-membrane associated rapid response steroid-binding receptor (1,25D3-MARRS receptor/ERp57). CONCLUSION: Diosgenin elicits L-type calcium channel-mediated extracellular Ca2+ influx, and thereby induces ADAM10-mediated ectodomain shedding of TNFR1. This effect of diosgenin was exerted through 1,25D3-MARRS receptor/ERp57.

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa.

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 µM) and a specified dye concentration (12 µM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules [Formula: see text] is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence.

Temporally resolved direct delivery of second messengers into cells using nanostraws.

Second messengers are biomolecules with the critical role of conveying information to intracellular targets. They are typically membrane-impermeable and only enter cells through tightly regulated transporters. Current methods for manipulating second messengers in cells require preparation of modified cell lines or significant disruptions in cell function, especially at the cell membrane. Here we demonstrate that 100 nm diameter 'nanostraws' penetrate the cell membrane to directly modulate second messenger concentrations within cells. Nanostraws are hollow vertical nanowires that provide a fluidic conduit into cells to allow time-resolved delivery of the signaling ion Ca(2+) without chemical permeabilization or genetic modification, minimizing cell perturbation. By integrating the nanostraw platform into a microfluidic device, we demonstrate coordinated delivery of Ca(2+) ions into hundreds of cells at the time scale of several seconds with the ability to deliver complex signal patterns, such as oscillations over time. The diffusive nature of nanostraw delivery gives the platform unique versatility, opening the possibility for time-resolved delivery of any freely diffusing molecules.

Calcium Uncaging with Visible Light.

We have designed a nitroaromatic photochemical protecting group that absorbs visible light in the violet-blue range. The chromophore is a dinitro derivative of bisstyrylthiophene (or BIST) that absorbs light very effectively (ε440 = 66,000 M(-1) cm(-1) and two-photon cross section of 350 GM at 775 nm). We developed a "caged calcium" molecule by conjugation of BIST to a Ca(2+) chelator that upon laser flash photolysis rapidly releases Ca(2+) in <0.2 ms. Using the patch-clamp method the optical probe, loaded with Ca(2+), was delivered into acutely isolated mouse cardiac myocytes, where either one- and two-photon uncaging of Ca(2+) induced highly local or cell-wide physiological Ca(2+) signaling events.