Fluorescence analysis of cells using a laser light source

A quantitative microfluorometric instrument is described that employs a helium cadmium laser (442 nm) as the illumination source. The instrument consists of a double grating monochromator in front of a gallium arsenide photomultiplier that is interfaced with a desktop computer. The versatility of the instrument in making quantitative nucleic acid measurements on acridine orange and Feulgen-Schiff stained cells is demonstrated. The ploidy levels of several populations are easily determined, and the Feulgen fluorescent emissions are considerably greater than those obtained with a standard mercury lamp.     

Spatio-temporal organization of receptive fields of the cat striate cortex

The responses of cortical cells to gratings and bars were compared. The excitatory and inhibitory on-and off-zones of a simple cell are composed of on- and off-subfields of CGL. Any zone is formed by an opponent pair of subfields one of which gives an excitatory effect, the other — inhibitory. Such organization assumes the linear properties of a simple field. The deviations from linearity are due to spatial dis-placements of the subfields, heterogeneity of subfields, or the absence of one subfield in the opponent pair. Subfields may be both phasic and tonic, even in the same RF. Analysis of the most common type of a complex cell with modulated responses against unmodulated background shows that a mask eliminating stimulation of any half of the RF causes (according to the theory of filtres) increasing the bandwidth due to the increase or the appearance of responses to side low and high frequencies. The modulated components of the responses from both halves of the RF are out of phase. Analysis of this fact and the responses to thin bars suggests that a complex field is formed by linear and nonlinear subsystems converging onto output neuron. Other types of complex fields are organized by different combinations of subsystems. Limited in area by masking the RF responds to much higher spatial frequencies than the whole RF. The optimal frequency in two-dimensional spatial frequency characteristics of the RF does not change with orientation. Simple RFs and a part of complex RF calculate the amplitude and the phase of the stimulus, the other part of complex RFs (with unmodulated response) calculate only amplitude. Given all this, the RFs are grating filters of spatial frequency.     

Promotion of double-strand break repair by human nuclear extracts preferentially involves recombination with intact homologous DNA.

Parameters of DNA double strand break (dsb) repair catalysed by human nuclear extract were analysed using, as substrate, the replicative form (RF) of M13 mp8 in which a single double strand break (dsb) was introduced by restriction. After incubation with the extract, the dsb repair was estimated by the ability of the incubated RF to produce plaques following transfection into JM 109 (Rec A-) bacteria. The possibility of recombination with a purified fragment from M13 mp8 RF enhances up to 20 times the plaquing ability of the RF. The repair by recombination occurs under several conditions: i) the break in the RF must be located in the region of homology with the fragment. ii) the fragment has to be intact in the region corresponding to the break in the RF. iii) a minimal length of homology between the region surrounding the dsb in the RF, and the fragment is required. The in vitro reaction is ATP dependent and dNTP's partially dependent. Dephosphorylation of the free ends in the RF decreases the repair by ligation but is without effect on the recombination.     

RF nonlinear interactions in living cells--II: detection methods for spectral signatures

The presence of harmonic products due to possible nonlinear interaction of amplitude modulated RF signals in living cells is best detected by using a cavity with high quality factor. Harmonic products generated by elementary oscillators can be trapped and accumulated in a cavity, permitting detection sensitivity much greater than in an open environment, where they would be radiated in all directions. The experimental method described herein is a systematic approach to detection of the non-Planck RF energy (if any) emitted by an exposed sample of living cells. Balzano and Sheppard [Balzano and Sheppard (2003): Bioelectromagnetics 24:473-482] classified the non-Planck RF emissions from living cells as coming from (1). nonlinear interactions and (2). inelastic interactions. Nonlinear harmonic products would appear in the band at twice the frequency of an amplitude modulated RF carrier. Inelastic interaction products resulting from the interaction between the incident RF energy and normally occurring mechanical vibrations are found in the band immediately adjacent to the carrier. Detection of the latter signals is difficult because of this close spectral proximity, for example, 1 part in 10(7) for 100 Hz modulation of a GHz carrier. Modern audio spectrum analyzers have excellent selectivity, providing 60 dB rejections only 2 kHz away from the carrier. By judicious selection of the amplitude modulation (AM) frequency, frequency of the RF carrier, and size of the biological sample, it is possible to achieve very high sensitivity (about -90 dBm) with commercially available instrumentation. The presence (or absence) of harmonics in the band adjacent to the amplitude modulated RF carrier would establish (or negate) the existence of coherent interactions between mechanical vibrations in the cell ensemble and the incident RF signal.     

Construction and evaluation of a frequency-domain epifluorescence microscope for lifetime and anisotropy decay measurements in subcellular domains

The measurement of time-resolved fluorescence parameters in living cells provides a powerful approach to study cell structure and dynamics. An epifluorescence microscope was constructed to resolve multi-component fluorescence lifetimes and complex anisotropy decay rapidly in labile biological samples. The excitation source consisted of focused, polarized laser light modulated by an impulse-driven Pockels' cell; parallel acquisition of phase angles and modulation amplitudes at more than 40 frequencies (5-250 MHz) was obtained by multi-harmonic cross-correlation detection. Lifetime decay was measured against standard solutions introduced into the light path proximal to the microscope objective. Anisotropy decay was measured by rotation of a Glan-Thompson polarizer in the emission path. Phase reference light was split from the beam proximal to the microscope. Optical components were selected to avoid depolarization and to optimize fluorescence detection efficiency. The dichoric was replaced by a 1 mm square mirror. Fitting routine statistics were optimized for model discrimination in realistic biological samples. Instrument performance was evaluated using fluorescein in H2O/glycerol and H2O/ethylene glycol mixtures and in Swiss 3T3 fibroblasts in monolayer culture. Objective depolarization effects were evaluated by measurement of anisotropy decay using objectives of different numerical aperture. Lifetime and anisotropy decay measured by microscopy (0.5 micron laser spot) agreed with data obtained by cuvette fluorimetry. New biological applications for time-resolved fluorescence microscopy are discussed.     

Review of possible modulation-dependent biological effects of radiofrequency fields

The biological effects of modulated radiofrequency (RF) electromagnetic fields have been a subject of debate since early publications more than 30 years ago, suggesting that relatively weak amplitude-modulated RF electromagnetic fields have specific biological effects different from the well-known thermal effects of RF energy. This discussion has been recently activated by the increasing human exposure to RF fields from wireless communication systems. Modulation is used in all wireless communication systems to enable the signal to carry information. A previous review in 1998 indicated that experimental evidence for modulation-specific effects of RF energy is weak. This article reviews recent studies (published after 1998) on the biological effects of modulated RF fields. The focus is on studies that have compared the effects of modulated and unmodulated (continuous wave) RF fields, or compared the effects of different kinds of modulations; studies that used only one type of signal are not included. While the majority of recent studies have reported no modulation-specific effects, there are a few interesting exceptions indicating that there may be specific effects from amplitude-modulated RF fields on the human central nervous system. These findings warrant follow-up studies.     

Cell oxidation–reduction imbalance after modulated radiofrequency radiation

Abstract

Aim of this study was to evaluate an influence of modulated radiofrequency field (RF) of 1800?MHz, strength of 30?V/m on oxidation–reduction processes within the cell. The assigned RF field was generated within Gigahertz Transversal Electromagnetic Mode cell equipped by signal generator, modulator, and amplifier. Cell line V79, was irradiated for 10, 30, and 60?min, specific absorption rate was calculated to be 1.6?W/kg. Cell metabolic activity and viability was determined by MTT assay. In order to define total protein content, colorimetric method was used. Concentration of oxidised proteins was evaluated by enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) marked with fluorescent probe 2′,7′-dichlorofluorescin diacetate were measured by means of plate reader device. In comparison with control cell samples, metabolic activity and total protein content in exposed cells did not differ significantly. Concentrations of carbonyl derivates, a product of protein oxidation, insignificantly but continuously increase with duration of exposure. In exposed samples, ROS level significantly (p?<?0.05) increased after 10?min of exposure. Decrease in ROS level was observed after 30-min treatment indicating antioxidant defence mechanism activation. In conclusion, under the given laboratory conditions, modulated RF radiation might cause impairment in cell oxidation–reduction equilibrium within the growing cells.     

Sensitive detection of small shifts in fluorescence spectra

Instrumentation is described for the sensitive detection of small shifts in the emission peaks of fluorescence spectra. The method is based on the modulation with time of the wavelength of the detected light at the emission maximum of the starting spectrum. Modulation of the wavelength is achieved by a novel use of a photoelastic light modulator and a specific arrangement of polarizing filters placed at the exit slit of the emission monochromator. A shift in the initial emission spectrum results in an alternating current in the detected light at double the fundamental frequency of the photoelastic modulator, which is detected with a phase-sensitive amplifier. The instrument is sensitive to shifts of 0.2 to 0.5 nm in the wavelength of maximal emission, verified with solutions of 5-dimethylaminonaphthalene-1-sulfonyl- -glutamic acid and reduced β-nicotinamide adenine dinucleotide by following fluorescence shifts resulting from alterations in solvent polarity. Also, the fluorescence of quinine (at micromolar levels) is detectable in the presence of a tenfold higher emission intensity of 9-aminoacridine, although the emission maxima of the two compounds are separated by less than 10 nm. The results of these experiments suggest applications of the technique to problems of biological interest which require sensitive detection of minute changes in fluorescence spectra, changes which are due to a shift in the emission spectrum of the chromophore studied or to the occurrence of a new emitting species which has a slightly shifted fluorescence spectrum.     

Allosteric aptamers controlling a signal amplification cascade allow visual detection of molecules at picomolar concentrations

A broadly applicable homogeneous detection system has been developed. It utilizes components of the blood coagulation cascade in the presence of polystyrene microspheres (MS) as a signal amplifier. Russell's viper venom factor X activator (RVV-X) triggers the cascade, which results in an eye-visible phase transition (precipitation) of MS bound to clotted fibrin. An allosteric RNA aptamer, RNA132, with affinity for RVV-X and human vascular endothelial growth factor (VEGF(165)) was created. RNA132 inhibits enzymatic activity of RVV-X. The effector molecule, VEGF(165), reverses the inhibitory activity of RNA132 on RVV-X and restores its enzymatic activity, thus, triggering the cascade and enabling the phase transition. As few as 5 fmol of VEGF(165) could be detected by the naked eye within an hour. Similar results were obtained for another allosteric aptamer modulated by a protein tyrosine phosphatase. The assay is instrumentation-free for both processing and readout and can be modified to detect molecules to which aptamers can be obtained.     

Correction of timing errors in photomultiplier tubes used in phase-modulation fluorometry

The measurement of fluorescence lifetimes is known to be hindered by the wavelenght-dependent and photocathode area-dependent time response of photomultiplier tubes. A simple and direct method is described to minimize the effects in photomultiplier tubes for phase-modulation fluorometry. Reference fluorophores of known lifetime were used in place of the usual scattering reference. The emission wavelenghts of the reference and sample were matched by either filters or a monochromator, and the use of a fluorophore rather than a scatter decreases the differences in spatial distribution of light emanating from the reference and sample. Thus photomultiplier tube artifacts are minimized. Five reference fluorophores were selected on the basis of availability, ease of solution preparation, and constancy of lifetime with temperature and emission wavelenght. These compounds are p-terphenyl, PPO, PPD, POPOP and dimethyl POPOP. These compounds are dissolved in ethanol to give standard solutions that can be used over the temperature range from ?55 to +55°C. Purging with inert gas is not necessary. The measured phase and modulation of the reference solution is used, in conjunction with the known reference, lifetime, to calculate the actual phase and modulation of the exictation beam. The use of standard fluorophores does not require separate experiments to quantify photomultiplier effects, and does not increase the time required for the measurement of fluorescence lifetimes. Examples are presented which demonstrate the elimination of artifactual photomultiplier effects in measurements of the lifetimes of DADH (0.4 ns) and indole solutions quenched by iodide. In addition, the use of these reference solutions increases the accuracy of fluorescence lifetime measurements ranging ranging to 30 ns. We judge this method to provide more reliable lifetime measurements by the phase and modulation method. The test solutions and procedures we describe may be used by other laboratories to evaluate the performance of their phase fluorometers.     

Phase precession through acceleration of local theta rhythm: a biophysical model for the interaction between place cells and local inhibitory neurons

Phase precession is one of the most well known examples within the temporal coding hypothesis. Here we present a biophysical spiking model for phase precession in hippocampal CA1 which focuses on the interaction between place cells and local inhibitory interneurons. The model's functional block is composed of a place cell (PC) connected with a local inhibitory cell (IC) which is modulated by the population theta rhythm. Both cells receive excitatory inputs from the entorhinal cortex (EC). These inputs are both theta modulated and space modulated. The dynamics of the two neuron types are described by integrate-and-fire models with conductance synapses, and the EC inputs are described using non-homogeneous Poisson processes. Phase precession in our model is caused by increased drive to specific PC/IC pairs when the animal is in their place field. The excitation increases the IC's firing rate, and this modulates the PC's firing rate such that both cells precess relative to theta. Our model implies that phase coding in place cells may not be independent from rate coding. The absence of restrictive connectivity constraints in this model predicts the generation of phase precession in any network with similar architecture and subject to a clocking rhythm, independently of the involvement in spatial tasks.     

High-performance liquid chromatography analysis of the thiobarbituric acid adducts of malonaldehyde and trans,trans-muconaldehyde

A reversed-phase HPLC method is described for the separation and analysis of the thiobarbituric acid (TBA) adducts of the reactive aldehydes muconaldehyde (MUC) and malonaldehyde (MDA). The TBA adduct of malonaldehyde was synthesized, purified, and its structure elucidated, for use as standard in quantitative HPLC studies. A detection limit of 1 X 10(-14) mol was achieved for the MUC:TBA and MDA:TBA adducts using the double monochromator fluorometric detector, 7 X 10(-13) mol was the detection limit using a variable wavelength uv-visible detector. Direct on-line identification of the eluting aldehyde:TBA adducts was achieved by the use of a diode-array uv-visible detector. The chromatographic behavior of the adducts under different mobile phase conditions was also examined. This HPLC methodology was used for the identification of muconaldehyde as a product of benzene oxidation in a hydroxyl radical generating system.     

Modulation of V1 Spike Response by Temporal Interval of Spatiotemporal Stimulus Sequence

The spike activity of single neurons of the primary visual cortex (V1) becomes more selective and reliable in response to wide-field natural scenes compared to smaller stimuli confined to the classical receptive field (RF). However, it is largely unknown what aspects of natural scenes increase the selectivity of V1 neurons. One hypothesis is that modulation by surround interaction is highly sensitive to small changes in spatiotemporal aspects of RF surround. Such a fine-tuned modulation would enable single neurons to hold information about spatiotemporal sequences of oriented stimuli, which extends the role of V1 neurons as a simple spatiotemporal filter confined to the RF. In the current study, we examined the hypothesis in the V1 of awake behaving monkeys, by testing whether the spike response of single V1 neurons is modulated by temporal interval of spatiotemporal stimulus sequence encompassing inside and outside the RF. We used two identical Gabor stimuli that were sequentially presented with a variable stimulus onset asynchrony (SOA): the preceding one (S1) outside the RF and the following one (S2) in the RF. This stimulus configuration enabled us to examine the spatiotemporal selectivity of response modulation from a focal surround region. Although S1 alone did not evoke spike responses, visual response to S2 was modulated for SOA in the range of tens of milliseconds. These results suggest that V1 neurons participate in processing spatiotemporal sequences of oriented stimuli extending outside the RF.     

SalB inactivation modulates culture supernatant exoproteins and affects autolysis and viability in Enterococcus faecalis OG1RF

The culture supernatant fraction of an Enterococcus faecalis gelE mutant of strain OG1RF contained elevated levels of the secreted antigen SalB. Using differential fluorescence gel electrophoresis (DIGE) the salB mutant was shown to possess a unique complement of exoproteins. Differentially abundant exoproteins were identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Stress-related proteins including DnaK, Dps family protein, SOD, and NADH peroxidase were present in greater quantity in the OG1RF salB mutant culture supernatant. Moreover, several proteins involved in cell wall synthesis and cell division, including d-Ala-d-Lac ligase and EzrA, were present in reduced quantity in OG1RF salB relative to the parent strain. The salB mutant displayed reduced viability and anomalous cell division, and these phenotypes were exacerbated in a gelE salB double mutant. An epistatic relationship between gelE and salB was not identified with respect to increased autolysis and cell morphological changes observed in the salB mutant. SalB was purified as a six-histidine-tagged protein to investigate peptidoglycan hydrolytic activity; however, activity was not evident. High-pressure liquid chromatography (HPLC) analysis of reduced muropeptides from peptidoglycan digested with mutanolysin revealed that the salB mutant and OG1RF were indistinguishable.     

Impairment of cobalt-induced riboflavin biosynthesis in a Debaryomyces hansenii mutant

Flavinogenic yeasts such as Debaryomyces hansenii overproduce riboflavin (RF) in the presence of heavy metals. Growth and RF production were compared between wild-type D. hansenii and a RF production-impaired metal-tolerant ura3 mutant in the presence of sublethal cobalt(II) concentrations. Debaryomyces hansenii (wild type) exhibits an extended lag phase with an increase in RF synthesis. Supplementation of exogenous uracil shortened the lag phase at the highest concentration of cobalt(II) used, suggesting that uracil has a possible role in metal acclimation. The D. hansenii ura3 mutant isolated by chemical mutagenesis exhibited a higher level of metal tolerance, no extended lag phase, and no marked increase in RF synthesis. Transformation of the mutant with the URA3 gene isolated from Saccharyomyces cerevisiae or D. hansenii did not restore wild-type characteristics, suggesting a second mutation that impairs RF oversynthesis. Our results demonstrate that growth, metal sensitivity, and RF biosynthesis are linked.     

Proposed test for detection of nonlinear responses in biological preparations exposed to RF energy

Demodulation of amplitude modulated radio frequency (RF) energy has been proposed as a mechanism for the biological responses to these fields. The experiment proposed here tests whether the electric and magnetic structures of biological cells exhibit the nonlinear responses necessary for demodulation. A high Q cavity and very low noise amplification can be used to detect ultraweak nonlinear responses that appear as a second harmonic of a RF field incident on the sample. Nonlinear fields scattered from metabolically active biological cells grown in monolayer or suspended in medium can be distinguished from nonlinearities of the apparatus. Estimates for the theoretical signal sensitivity and analysis of system noise indicate the possibility of detecting a microwave signal at 1.8 GHz (2nd harmonic of 900 MHz) as weak as one microwave photon per cell per second. The practical limit, set by degradation of the cavity Q, is extremely low compared to the much brighter thermal background, which has its peak in the infrared at a wavelength of about 17 microm and radiates 10(10) infrared photons per second per cell in the narrow frequency band within 0.5% of the peak. The system can be calibrated by introduction of known quantities of nonlinear material, e.g., a Schottky diode. For an input power of 160 microW at 900 MHz incident on such biological material, the apparatus is estimated to produce a robust output signal of 0.10 mV at 1.8 GHz if detected with a spectrum analyzer and a 30-dB gain low noise amplifier. The experimental threshold for detection of nonlinear interaction phenomena is 10(10) below the signal produced by a Schottky diode, giving an unprecedented sensitivity to the measurement of nonlinear energy conversion processes in living tissue.     

Evaluation of a technique for the measurement of chlorophyll fluorescence from leaves exposed to continuous white light

Abstract An instrument for the generation and measurement of modulated chlorophyll fluorescence signals from leaves exposed to continuous, highintensity white light is described. Modulated fluorescence is generated in the leaf by pulsed diodes emitting low-intensity yellow radiation and is detected with a photodiode whose output is fed to an amplifier locked in to the frequency of the lightemitting diodes. Comparisons are made between the modulated fluorescence signals measured with this instrument and the continuous fluorescence signals emitted from dark-adapted leaf tissue and isolated thylakoids when photosynthetic activity is induced by exposure to a range of intensities of continuous broad-band, blue-green light. The modulated fluorescence signals were similar to the continuous fluorescence signals, but they were not always identical. The small differences between the two signals are mainly attributable to differences in the populations of chloroplasts being monitored in the two measurements as a result of differential penetration of the modulated and actinic light sources into the sample.     

Apoptosis induced by ultraviolet radiation is enhanced by amplitude modulated radiofrequency radiation in mutant yeast cells

The aim of this study was to investigate whether radiofrequency (RF) electromagnetic field (EMF) exposure affects cell death processes of yeast cells. Saccharomyces cerevisiae yeast cells of the strains KFy417 (wild-type) and KFy437 (cdc48-mutant) were exposed to 900 or 872 MHz RF fields, with or without exposure to ultraviolet (UV) radiation, and incubated simultaneously with elevated temperature (+37 degrees C) to induce apoptosis in the cdc48-mutated strain. The RF exposure was carried out in a special waveguide exposure chamber where the temperature of the cell cultures can be precisely controlled. Apoptosis was analyzed using the annexin V-FITC method utilizing flow cytometry. Amplitude modulated (217 pulses per second) RF exposure significantly enhanced UV induced apoptosis in cdc48-mutated cells, but no effect was observed in cells exposed to unmodulated fields at identical time-average specfic absorption rates (SAR, 0.4 or 3.0 W/kg). The findings suggest that amplitude modulated RF fields, together with known damaging agents, can affect the cell death process in mutated yeast cells. Bioelectromagnetics 25:127-133, 2004.     

Regulation of eukaryotic protein synthesis by protein kinases that phosphorylate initiation factor eIF-2: Further evidence for a common mechanism of inhibition of protein synthesis

The role of reversing factor (RF) in the regulation of protein synthesis by inhibitory protein kinases that phosphorylate the 38,000-dalton subunit of initiation factor eIF-2 has been examined. Results show that as with the heme-regulated protein kinase (HRI), RF restores protein synthesis in reticulocyte lysates inhibited by translational inhibitors from rat liver, wheat germ, Krebs ascites cell, by oxidized glutathione, the protein kinase activated by double stranded RNA (dRI), and the interferon-induced double stranded RNA activated protein kinase from Ehrlich ascites and Hela cells. These findings suggest that RF plays an important role in eukaryotic protein chain initiation cycle.     

Toward a Better Raft Model: Modulated Phases in the Four-Component Bilayer,DSPC/DOPC/POPC/CHOL

The liquid-liquid (Ld + Lo) coexistence region within a distearoyl-phosphatidylcholine/dioleoyl-phosphatidylcholine/palmitoyl-oleoyl-phosphatidylcholine/cholesterol (DSPC/DOPC/POPC/CHOL) mixture displays a nanoscopic-to-macroscopic transition of phase domains as POPC is replaced by DOPC. Previously, we showed that the transition goes through a modulated phase regime during this replacement, in which patterned liquid phase morphologies are observed on giant unilamellar vesicles (GUVs). Here, we describe a more detailed investigation of the modulated phase regime along two different thermodynamic tielines within the Ld + Lo region of this four-component mixture. Using fluorescence microscopy of GUVs, we found that the modulated phase regime occurs at relatively narrow DOPC/(DOPC+POPC) ratios. This modulated phase window shifts to higher values of DOPC/(DOPC+POPC) when CHOL concentration is increased, and coexisting phases become closer in properties. Monte Carlo simulations reproduced the patterns observed on GUVs, using a competing interactions model of line tension and curvature energies. Sufficiently low line tension and high bending moduli are required to generate stable modulated phases. Altogether, our studies indicate that by tuning the lipid composition, both the domain size and morphology can be altered drastically within a narrow composition space. This lends insight into a possible mechanism whereby cells can reorganize plasma membrane compartmentalization simply by tuning the local membrane composition or line tension.