Changes In Relative Fluorescence Research Articles

Point-of-Care Molecular Testing with tinto rangTM: A Food Grade Safe Fluorophore for Colorimetric LAMP Assays at Low Resource Settings

LAMP (loop-mediated isothermal amplification) has proven to be a highly robust, sensitive, and cost-effective method for detecting specific target nucleic acid sequences in a single-tube reaction without the need for sophisticated instruments. Traditionally, pH-sensitive dyes, DNA-binding dyes like Ethidium Bromide(EtBr), and SYBR have been employed to detect amplified products. However, these dyes present several drawbacks, limiting the widespread adoption of LAMP in diagnostic applications. To address this limitation, a novel dye called tinto rangTM has been introduced with promising applications in nucleic acid analysis. In this study, we present an evaluation of tinto rangTM's performance in comparison to commercially available dyes commonly used in LAMP assays. One of the unique advantages of tinto rangTM is its capability to facilitate the analysis of amplified nucleic acid products through three distinct methods: visual color change, changes in relative fluorescence, and turbidity. In addition to assessing tinto rangTM's performance, we also investigated various approaches to tackle the issue of non-specific amplification, which has been a common challenge in LAMP reactions. The experimental results unequivocally demonstrate that tinto rangTM outperforms pH-indicating and other DNA-binding dyes used in LAMP assays, making it a superior alternative. This study paves the way for the broader adoption of tinto rangTM in various applications, including point-of-care diagnostics, disease monitoring, and molecular biology research.

Point-of-Care Molecular Testing with tinto rangTM: A Food Grade Safe Fluorophore for Colorimetric LAMP Assays at Low Resource Settings

LAMP (loop-mediated isothermal amplification) has proven to be a highly robust, sensitive, and cost-effective method for detecting specific target nucleic acid sequences in a single-tube reaction without the need for sophisticated instruments. Traditionally, pH-sensitive dyes, DNA-binding dyes like Ethidium Bromide(EtBr), and SYBR have been employed to detect amplified products. However, these dyes present several drawbacks, limiting the widespread adoption of LAMP in diagnostic applications. To address this limitation, a novel dye called tinto rangTM has been introduced with promising applications in nucleic acid analysis. In this study, we present an evaluation of tinto rangTM's performance in comparison to commercially available dyes commonly used in LAMP assays. One of the unique advantages of tinto rangTM is its capability to facilitate the analysis of amplified nucleic acid products through three distinct methods: visual color change, changes in relative fluorescence, and turbidity. In addition to assessing tinto rangTM's performance, we also investigated various approaches to tackle the issue of non-specific amplification, which has been a common challenge in LAMP reactions. The experimental results unequivocally demonstrate that tinto rangTM outperforms pH-indicating and other DNA-binding dyes used in LAMP assays, making it a superior alternative. This study paves the way for the broader adoption of tinto rangTM in various applications, including point-of-care diagnostics, disease monitoring, and molecular biology research.

Pecularities of reactive oxygen species accumulation in the primary culture of nerve cells of Lymnaea stagnalis

A fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate was used to characterize an accumulation of reactive oxygen species (ROS) in the primary culture (72 and 144 h) of neurons of the central ganglia of the mollusc Lymnaea stagnalis under conditions of acute, induced oxidative stress. It was found that larger cells accumulate a greater amount of ROS. High values of the relative (per unit area) fluorescence, indicating an increase in the amount of ROS per unit of intracellular space, are characteristic of smaller neurons of 72-hour culture. Changes in relative fluorescence over time are different from each other in neurons of 72- and 144-hour cultures. For an older culture, a negative, statistically significant relationship was noted (R = – 0.31), and for cells of 72 h of cultivation, a statistically significant correlation was not found (R = 0.12). It is assumed that the discovered relationship between the size (shape) of a neuron and its ability to resist the accumulation of ROS in the cytosol indicates differences in the resistance of CNS neurons to oxidative damage depending on their place and position in the neural network, thereby ensuring the selective stability of neuronal functions under conditions of oxidative stress.

Isosbestic Point in Optical Mapping; Theoretical and Experimental Determination With Di-4-ANBDQPQ Transmembrane Voltage Sensitive Dye.

Optical mapping methods utilize fluorescence dyes to measure dynamic response of cardiac tissue such as changes in transmembrane potential (Vm). For the commonly used Vm sensitive dyes, a dye absorption and emission spectra shift as Vm changes. Signals relevant to Vm are calculated as a relative fluorescence change with respect to the fluorescence baseline. The amplitude of the change depends on the long-pass (LP) filter cut-on wavelength, placed on the sensor side, and the excitation wavelength. An excitation wavelength near the absorption peak, termed the isosbestic point, results in minimal absorption coefficient change as absorption spectra shifts. Consequentially the fluorescence intensity virtually does not change, when fluorescence across the entire emission spectra is measured, irrelevant of Vm changes. In this study we experimentally determined the isosbestic point for a near infrared dye Di-4-ANBDQPQ. We then present a theoretical study examining the dye linear or non-linear response as the fractional fluorescence change of Vm change, due to emission spectra shift and amplitude change, over a range of excitation wavelengths and LP filters. Linear "optical" response is important to quantify certain aspects of cardiac dynamics such as the action potential (AP) shape and duration, especially when studying drug effects and dynamical substrates for arrhythmia development.

Influence of SiO2 nanoparticles on relative fluorescence of plant cells

Nanoparticles (nano-scale particles (NSPs)) are defined as particles with dimensions less than 100 nm. SiO<sub>2</sub> nanoparticles are one of the most widely common nanoparticles in the environment, particularly in urban areas. The sources of SiO<sub>2 </sub>nanoparticles are very different, including natural nanoparticles, anthropogenic and engineered nanoparticles. The SiO<sub>2 </sub>nanoparticles could be considered a source of different pollution effects on leaving organisms. Nevertheless, knowledge of the mechanisms, through which the SiO<sub>2 </sub>nanoparticles affect cells, is incomplete. The aim of the research was to elaborate a method to determine changes in relative fluorescence of both somatic and immature gametic plant cells in presence of SiO<sub>2</sub> nanoparticles. Relative cell fluorescence was measured with BD FACSJazz® cell sorter using 488 nm exciting laser light. Mean cell fluorescence was determined for samples of purified cell suspension. Gates of different size and shape were preliminary tested to find those with the lowest CV. Cell plots were created by BS FACS Software 1.0.0.650. The densest part of the plot was gated using oval-shaped gate. The gate included 95-99% of all cells. A logarithmic scale in arbitrary fluorescence units was applied to determine cell relative fluorescence. More than 10 000 cells were gated and analysed from each sample. Somatic cell culture from callus culture initiated from leaves of flax (<em>Linum usitatissimum) </em>was obtained. The relative fluorescence of the somatic cells had large distribution, since the cells differ by many parameters (size, shape, metabolism etc.). Immature pollen cells (one-nucleus stage) as best for SiO<sub>2</sub> nanoparticles influence investigation were found. The influence of SiO<sub>2</sub> nanoparticles on several plant species (<em>Cyclamen persicum, Tilia cordata, Hordeum vulgare</em> and <em>Triticum </em><em>aestivum</em>) immature pollen cells were investigated. A significant increase in relative cell fluorescence was observed for all mentioned plant species cells after incubation in SiO<sub>2</sub> nanoparticles suspension. It was found that cell relative fluorescence was dependent on cultivation duration in SiO<sub>2</sub> nanoparticles suspension.

An Interaction of Oxytocin Receptors with Metabotropic Glutamate Receptors in Hypothalamic Astrocytes

Hypothalamic astrocytes play a critical role in the regulation and support of many different neuroendocrine events, and are affected by oestradiol. Both nuclear and membrane oestrogen receptors (ERs) are expressed in astrocytes. Upon oestradiol activation, membrane-associated ER signals through the type 1a metabotropic glutamate receptor (mGluR1a) to induce an increase of free cytoplasmic calcium concentration ([Ca(2+)](i)). Because the expression of oxytocin receptors (OTRs) is modulated by oestradiol, we tested whether oestradiol also influences oxytocin signalling. Oxytocin at 1, 10, and 100 nm induced a [Ca(2+)](i) flux measured as a change in relative fluorescence [DeltaF Ca(2+) = 330 +/- 17 relative fluorescent units (RFU), DeltaF Ca(2+) = 331 +/- 22 RFU, and DeltaF Ca(2+) = 347 +/- 13 RFU, respectively] in primary cultures of female post-pubertal hypothalamic astrocytes. Interestingly, OTRs interacted with mGluRs. The mGluR1a antagonist, LY 367385 (20 nm), blocked the oxytocin (1 nm)-induced [Ca(2+)](i) flux (DeltaF Ca(2+) = 344 +/- 19 versus 127 +/- 11 RFU, P < 0.001). Conversely, the mGluR1a receptor agonist, (RS)-3,5-dihydroxyphenyl-glycine (100 nm), increased the oxytocin (1 nm)-induced [Ca(2+)](i) response (DeltaF Ca(2+) = 670 +/- 31 RFU) compared to either compound alone (P < 0.001). Because both oxytocin and oestradiol rapidly signal through the mGluR1a, we treated hypothalamic astrocytes sequentially with oxytocin and oestradiol to determine whether stimulation with one hormone affected the subsequent [Ca(2+)](i) response to the second hormone. Oestradiol treatment did not change the subsequent [Ca(2+)](i) flux to oxytocin (P > 0.05) and previous oxytocin exposure did not affect the [Ca(2+)](i) response to oestradiol (P > 0.05). Furthermore, simultaneous oestradiol and oxytocin stimulation failed to yield a synergistic [Ca(2+)](i) response. These results suggest that the OTR signals through the mGluR1a to release Ca(2+) from intracellular stores and rapid, nongenomic oestradiol stimulation does not influence OTR signalling in astrocytes.

Membrane Estrogen Receptor-α Interacts with Metabotropic Glutamate Receptor Type 1a to Mobilize Intracellular Calcium in Hypothalamic Astrocytes

Estradiol, acting on a membrane-associated estrogen receptor-alpha (mERalpha), induces an increase in free cytoplasmic calcium concentration ([Ca(2+)](i)) needed for progesterone synthesis in hypothalamic astrocytes. To determine whether rapid estradiol signaling involves an interaction of mERalpha with metabotropic glutamate receptor type 1a (mGluR1a), changes in [Ca(2+)](i) were monitored with the calcium indicator, Fluo-4 AM, in primary cultures of female postpubertal hypothalamic astrocytes. 17beta-Estradiol over a range of 1 nm to 100 nm induced a maximal increase in [Ca(2+)](i) flux measured as a change in relative fluorescence [DeltaF Ca(2+) = 615 +/- 36 to 641 +/- 47 relative fluorescent units (RFU)], whereas 0.1 nm of estradiol stimulated a moderate [Ca(2+)](i) increase (275 +/- 16 RFU). The rapid estradiol-induced [Ca(2+)](i) flux was blocked with 1 microm of the estrogen receptor antagonist ICI 182,780 (635 +/- 24 vs. 102 +/- 11 RFU, P < 0.001) and 20 nmof the mGluR1a antagonist LY 367385 (617 +/- 35 vs. 133 +/- 20 RFU, P < 0.001). Whereas the mGluR1a receptor agonist (RS)-3,5-dihydroxyphenyl-glycine (50 microm) also stimulated a robust [Ca(2+)](i) flux (626 +/- 23 RFU), combined treatment of estradiol (1 nm) plus (RS)-3,5-dihydroxyphenyl-glycine (50 microm) augmented the [Ca(2+)](i) response (762 +/- 17 RFU) compared with either compound alone (P < 0.001). Coimmunoprecipitation demonstrated a direct physical interaction between mERalpha and mGluR1a in the plasma membrane of hypothalamic astrocytes. These results indicate that mERalpha acts through mGluR1a, and mGluR1a activation facilitates the estradiol response, suggesting that neural activity can modify estradiol-induced membrane signaling in astrocytes.

Characterization of Molecular Interactions between Eosinophil Cationic Protein and Heparin

Eosinophil cationic protein (ECP) is currently used as a biomarker for airway inflammation. It is a heparin-binding ribonuclease released by activated eosinophils. Its cytotoxicity toward cancer cell lines is blocked by heparin. The objective of this study was to locate the heparin binding site of ECP by site-directed mutagenesis and construction of a synthetic peptide derived from this region. Synthetic heparin with > or =5 monosaccharide units showed strong inhibition of ECP binding to the cell surface. Analysis of ECP mt1 (R34A/W35A/R36A/K38A) showed that these charged and aromatic residues were involved in ECP binding to heparin and the cell surface. A potential binding motif is located in the loop L3 region between helix alpha2 and strand beta1, outside the RNA binding domain. The synthetic peptide derived from the loop L3 region displayed strong pentasaccharide binding affinity and blocked ECP binding to cells. In addition, ECP mt1 showed reduced cytotoxicity. Thus, the tight interaction between ECP and heparin acts as the primary step for ECP endocytosis. These results provide new insights into the structure and function of ECP for anti-asthma therapy.

Enhanced function with decreased internalization of carboxy-terminus truncated CXCR4 responsible for WHIM syndrome

WHIM (warts, hypogammaglobulinemia, recurrent bacterial infection, myelokathexis) syndrome is an autosomal dominant immune deficiency with severe chronic neutropenia and marrow neutrophil apoptosis. Carboxy-termini truncating mutations in the chemokine receptor CXCR4 have been identified in WHIM patients. We created a retrovirus encoding mutated CXCR4 (truncating point mutation 1000C-->T [R334X] inherited heterozygously in several WHIM patients) in order to transducer healthy human CD34 stem cells and K562 to overexpress mutated CXCR4 and determined its effect on receptor responses to stromal-derived factor-1 (SDF1). Retrovirus vector was engineered to coexpress WHIM-associated R334X mutated CXCR4 together with green fluorescent protein (GFP). Control vectors included similar constructs with wild-type CXCR4 (WT-CXCR4) or only GFP. CD34+ cells and K562 were transduced with these vectors. Populations of 100% transduced K562 were established by sorting GFP+ cells by flow cytometry. We performed migration and calcium flux assays of transduced CD34+ cells and transduced/sorted K562. We also examined receptor recycling in response to SDF1. Healthy human CD34+ cells and/or human erythroleukemia K562 cells transduced to express mutated CXCR4, WT-CXCR4, or GFP alone demonstrated that mutated CXCR4 was associated with enhanced calcium flux and enhanced migration. There was also decreased receptor internalization and enhanced recovery of surface mutated CXCR4 in response to SDF1 compared with WT-CXCR4. We propose that decreased internalization of WHIM-associated mutated CXCR4 leads to prolongation/enhancement of signaling in response to SDF1 and that this may provide the biochemical basis for the autosomal dominant abnormalities of cell trafficking and function associated with WHIM syndrome.

Possible sources of dye-related signal correlation bias in two-color DNA microarray assays

DNA microarray analyses commonly use two spectrally distinct fluorescent labels to simultaneously compare different mRNA pools. Signal correlation bias currently limits accepted resolution to twofold changes in gene expression. This bias was investigated by (i) examining fluorescence and absorption spectra and changes in relative fluorescence of DNAs labeled with the Cy3, Cy5, Alexa Fluor ® 555, and Alexa Fluor 647 dyes and by (ii) using homotypic hybridization assays to compare the Cy dye pair with the Alexa Fluor dye pair. Cy3 or Cy5 dye-labeled DNA exhibited reduced fluorescence and absorption anomalies that were eliminated by nuclease treatment, consistent with fluorescence quenching that arises from dye–dye or dye–DNA–dye interactions. Alexa Fluor 555 and Alexa Fluor 647 dye-labeled DNA exhibited little or no such anomalies. In microarray hybridization, the Alexa Fluor dye pair provided higher signal correlation coefficients ( R 2) than did the Cy dye pair; at the 95% prediction level, a 1.3-fold change in gene expression was significant using the Alexa Fluor dye pair. Lowered signal correlation of the Cy dye pair was associated with high variance in Cy5 dye signals. These results indicate that fluorescence quenching may be a source of signal bias associated with the Cy dye pair.

Mechanism linking NMDA receptor activation to modulation of voltage-gated sodium current in distal retina.

In this study, we investigated the mechanism that links activation of N-methyl-D-aspartate (NMDA) receptors to inhibition of voltage-gated sodium channels in isolated catfish cone horizontal cells. NMDA channels were activated in voltage-clamped cells incubated in low-calcium saline or dialyzed with the calcium chelator BAPTA to determine that calcium influx through NMDA channels is required for sodium channel modulation. To determine whether calcium influx through NMDA channels triggers calcium-induced calcium release (CICR), cells were loaded with the calcium-sensitive dye calcium green 2 and changes in relative fluorescence were measured in response to NMDA. Responses were compared with measurements obtained when caffeine depleted stores. Voltage-clamp studies demonstrated that CICR modulated sodium channels in a manner similar to that of NMDA. Blocking NMDA receptors with AP-7, blocking CICR with ruthenium red, depleting stores with caffeine, or dialyzing cells with calmodulin antagonists W-5 or peptide 290-309 all prevented sodium channel modulation. These results support the hypothesis that NMDA modulation of voltage-gated sodium channels in horizontal cells requires CICR and activation of a calmodulin-dependent signaling pathway.

Evaluation of Red Morningglory (Ipomoea coccinea) for Potential Atrazine Resistance1

Laboratory and greenhouse studies were conducted to determine if the reported red morningglory control failures with atrazine in sugarcane are caused by triazine-resistant mutants. Plants were grown from seeds collected at 24 locations in Louisiana and Arkansas where atrazine had never been used, and where it had been used in sugarcane with poor results. Terminal fluorescence of leaf material from all locations increased after treatment with 10−3 M atrazine, indicating electron transport inhibition and, hence, triazine susceptibility. However, small differences in the magnitude of fluorescence increase were observed among populations, possibly indicating the existence of biotypes with slightly different inherent tolerances to atrazine. Some phenotypic differences were observed among the red morningglory populations. In a separate study, postemergence application of atrazine at 1.1 kg ai/ha plus nonionic surfactant controlled greenhouse-grown plants from all populations at least 99%, which supports the findings of the fluorescence assay. This research was unable to verify that reduced red morningglory control with atrazine was the result of an altered binding site mutation, even in populations exposed to atrazine annually for more than 10 yr. Other factors should be evaluated to determine their impact on atrazine performance.Nomenclature: Atrazine; red morningglory, Ipomoea coccinea L. #3 IPOCC; sugarcane, Saccharum interspecific hybrids.Additional index words: Chlorophyll fluorescence, herbicide resistance, IPOCC, photosynthesis inhibition, photosystem II, triazine resistance, weed biotypes.Abbreviations: CRF, change in relative fluorescence; DAT, days after treatment; FT, terminal fluorescence; POST, postemergence; PRE, preemergence.

The mammalian profilin isoforms display complementary affinities for PIP2 and proline-rich sequences.

We present a study on the binding properties of the bovine profilin isoforms to both phosphatidylinositol 4,5-bisphosphate (PIP2) and proline-rich peptides derived from vasodilator-stimulated phosphoprotein (VASP) and cyclase-associated protein (CAP). Using microfiltration, we show that compared with profilin II, profilin I has a higher affinity for PIP2. On the other hand, fluorescence spectroscopy reveals that proline-rich peptides bind better to profilin II. At micromolar concentrations, profilin II dimerizes upon binding to proline-rich peptides. Circular dichroism measurements of profilin II reveal a significant conformational change in this protein upon binding of the peptide. We show further that PIP2 effectively competes for binding of profilin I to poly-L-proline, since this isoform, but not profilin II, can be eluted from a poly-L-proline column with PIP2. Using affinity chromatography on either profilin isoform, we identified profilin II as the preferred ligand for VASP in bovine brain extracts. The complementary affinities of the profilin isoforms for PIP2 and the proline-rich peptides offer the cell an opportunity to direct actin assembly at different subcellular localizations through the same or different signal transduction pathways.

Fluorescence and circular dichroism studies during the interactions of sulfated polysaccharides with antithrombin III

The changes in relative fluorescence of antithrombin III (AT-III) during its interaction with sulfated xylans were compared with that of sulfated glycosaminoglycans by measuring the ratio of the increase in fluorescence of AT-III in the presence of sulfated polysaccharide to the fluorescence of AT-III alone for various mass ratios. Interactions of corn cob xylan sulfate (CCXS) and sodium pentosan polysulfate(SP-54) with AT-III resulted in enhancements of relative fluorescence which were lower than commercial heparin. At mass ratios below 1, heparan sulfate and low molecular weight heparin (LMWH) gave increases in the relative fluorescence higher than that of commercial heparin, while highly sulfated semisynthetic chondroitin sulfates A and C gave much smaller increases. The relative fluorescence enhancements of AT-III by heparan sulfate, commercial heparin, LMWH and heparin derived pentasaccharide (HDP) increased with increasing mass ratios while the enhancements by CCXS, SP-54 and the highly sulfated chondroitin sulfates A and C were reversed at higher mass ratios. The estimated dissociation constants (k d) for the interaction of AT-III and the heparin-related compounds showed that heparan sulfate and LMWH gave the lowest k d values indicating a higher affinity for AT-III while commercial heparin and HDP gave higher k d values, indicating a lower affinity for AT-III. SP-54 gave a k d value lower than CCXS, indicating a greater affinity for AT-III. A comparison of the near ultraviolet (UV) circular dichroism (CD) spectrum of ATIII alone and during its interaction with oat spelts xylan sulfate (OSXS) showed enhancements of the two aromatic amino acid regions corresponding to phenylalanine and tryptophan.

Glutamate-induced deporalization in earthworm ventral nerve cord

We investigated glutamate-induced neuron response in the ventral nerve cord of the earthworm ( Eisenia foetida) using a voltage-sensitive dye-imaging technique. Isolated earthworm ganglia were stained by fluorescence voltage-sensitive dye, RH414, and voltage image was acquired by a conventional charge-coupled device (CCD) technique or a confocal laser scanning microscope. The fluorescence images before and during the glutamate stimulation was acquired and the relative fluorescence change was imaged as pseudo-color. Bath-applied glutamate (1 mM) depolarized many neurons on the ventral side, and in the three giant fibers on the dorsal side.

Changes in membrane potential of intact adipocytes measured with fluorescent dyes

The change in transmembrane potential of rat adipocytes was measured using the fluorescent probes 3,3′-diethylthiadicarbocyanine iodide, [di-SC 2(5)], and 3,3′-dipropylthiadicarbocyanine iodide, [di-SC 3(5)]. The fluorimetric technique was calibrated by altering the potassium ion concentration while keeping the sum of potassium and sodium ions at a constant concentration of 153 mM. A 3% change in relative fluorescence was equivalent to a membrane potential change of 1 mV. This method was used to measure changes in transmembrane potential by insulin and two insulin-mimetic agents: wheat germ agglutinin and ethanolamine.

Changes in membrane potential of intact adipocytes measured with fluorescent dyes

The change in transmembrane potential of rat adipocytes was measured using the fluorescent probes 3,3′-diethylthiadicarbocyanine iodide, [di-SC 2(5)], and 3,3′-dipropylthiadicarbocyanine iodide, [di-SC 3(5)]. The fluorimetric technique was calibrated by altering the potassium ion concentration while keeping the sum of potassium and sodium ions at a constant concentration of 153 m M. A 3% change in relative fluorescence was equivalent to a membrane potential change of 1 mV. This method was used to measure changes in transmembrane potential by insulin and two insulin-mimetic agents: wheat germ agglutinin and ethanolamine.

Genetic Variability for Seedling Atrazine Tolerance in Indiangrass

Indiangrass [Sorghastrum nutans (L.) Nash] lacks sufficient seedling atrazine [6‐chloro‐N‐ethyl‐N′‐(1‐methylethyl)‐l,3,5‐triazine‐2,4‐diamine] tolerance to permit the use of atrazine as a pre‐emergence herbicide. The principle objective of this study was to estimate the genetic variability for seedling atrazine tolerance in two indiangrass populations, ‘Nebraska 54’ and ‘Holt’, using seed harvested from replicated clones. Seedling survival in soil containing 3 mg kg−1 atrazine was determined in the greenhouse. There was significant genetic variability among half‐sib families of both populations for seedling atrazine tolerance. Heritability estimates were greater than 0.50. There was also significant genetic variability among the parent plants of both populations for atrazine tolerance as measured by the change in relative fluorescence (CRF) of leaf disks exposed to atrazinc. Atrazine blocks photosynthesis, causing differential fluorescence among plants differing in tolerance, which can be measured with a fluorometer. Correlations of CRF of the parent plants and the seedling atrazine tolerance of their progeny were low (r &lt; 0.3) indicating that fluorescence assay would not be a usable screening procedure. Improving the seedling atrazine tolerance of indiangrass using the greenhouse soil test and conventional breeding methods is possible.

Membrane potential induced by external electric field pulses can be followed with a potentiometric dye

A potential-sensitive dye was recently used to measure the spatial variation in the membrane potential induced by an externally applied electric field. In this work, we demonstrate that the time course of these induced potentials can also be followed. Two experimental systems were explored. Dye fluorescence from HeLa cells could be modulated by a train of field pulses; the relative fluorescence change measured with a lock-in amplifier was linear with the field and similar to the fluorescence responses obtained in the static measurements. A model membrane system consisting of a hemispherical bilayer allowed convenient measurement of the dye absorbance change as a function of the bathing solution conductivity. The charging time of the membrane was inversely related to the aqueous conductance as predicted by the theoretical solution to Laplace's equation.

Magnetic field affects the fluorescence yield in reaction center preparations from Rhodopseudomonas sphaeroides R-26

Purified photochemical reaction centers from Rhodopseudomonas sphaeroides R-26 were reduced with Na 2S 2O 4 so as to block their photochemical electron-transfer reactions. The magnetic field induced an increase in the emission yield. Our results support the hypothesis that under these conditions, charge recombination in the singlet radical pair composed of the oxidized primary donor and reduced primary acceptor predominantly generates the excited singlet state of the reaction center bacteriochlorophyll. The maximum relative fluorescence change and the value of the magnetic field at which half-saturation of the effect is achieved ( B 1 2 ) at room temperature are 5.5% and 75 G, respectively. For the whole cells of Rps. sphaeroides R-26 these parameters are 1.2% and 120 G. The relative fluorescence change at 600 G, ΔF F(600) , and B 1 2 are studied as functions of temperature. The temperature dependencies of ΔF F(600) for reaction centers and whole cells of Rps. sphaeroides R-26 are qualitatively the same, with the maximum effect (8% for reaction centers) occurring at 230 K. However, the B 1 2 curves for the two preparations are different.