Phase-sensitive Detection Method Research Articles

Application of polarized modulation technique in polymer science

The material is presented for those who intend to use polarization modulation and phase-sensitive detection methods in their work. The know-how is presented on optical and electric components used in polarization-modulation instruments. Three essentials in polarization-modulation spectroscopy-the Mueller matrix approach, the photoelastic modulator (PEM), and the phase-sensitive detection method-are intensively discussed. How versatile and powerful the Mueller matrix approach is in the analysis, evaluation, and understanding of problems associated with polarization-modulation measurements is shown. Use of the Mueller matrix approach in the interpretation and design of experiments in polarization modulation spectroscopy is recommended

Delay in granular fusion evoked by repetitive cytosolic Ca 2+ spikes in mouse pancreatic acinar cells

Patch-clamp whole-cell recording in combination with a phase-sensitive detection method was applied to single, enzymatically isolated, mouse pancreatic acinar cells. Either muscarinic stimulation with a low concentration of ACh (50 nM) or cell infusion of inositol 1,4,5-trisphosphate (InsP 3) induced repetitive spike-like increases of membrane capacitance (ΔC), membrane conductance (ΔG) and membrane current (ΔI). Cellular perfusion of InsP 3, 10 μM in patch-pipettes, induced baseline spikes in ΔC and ΔG, resembling those evoked by ACh. The result indicates that exocytotic granular fusion is primarily triggered by the InsP 3-induced repetitive rise of [Ca 2+] i. The ACh-induced ΔC took off almost synchronously with ΔG with an apparent delay of less than 40 ms in the initial spike response. This delay of ΔC, however, becomes longer by a factor of 7–12 during repetitive Ca 2+ spike cycles. Concomitantly a faster decrease in ΔC spikes than ΔG spikes was observed during the cycles. Two explanations are proposed. First, the Ca 2+ sensitivity of granular fusion decreases during the repetitive Ca 2+ spikes. This might be due to gradual washout of low molecular components responsible for exocytosis under the whole-cell recording condition. Second, the pool of immediately releasable or of primed zymogen granules is easily exhausted or desensitized during the Ca 2+ spike cycles, and has to be supplied from newly primed or sensitized resources. The progressive delay in ΔC during the spike cycle is interpreted as a delay in the process of supplying fusible granules.

Chapter 36 Phase-Sensitive Detection Methods for Resolving Fluorescence Emission Signals and Directly Quantifying Lifetime

This chapter discusses phase-sensitive detection methods for resolving fluorescence emission signals and directly quantifying lifetime. A new flow cytometry (FCM) approach, based on phase-resolved fluorescence spectroscopy methods that provides unique capabilities for separating signals from multiple overlapping emissions in fluorochrome-labeled cells as they pass across a modulated excitation source, has been developed. The measurement of fluorescence lifetime also is of importance because it provides additional information about fluorochrome/cell interactions. An important advantage of lifetime measurements is that lifetimes in some cases can be considered as absolute quantities. However, the lifetime of dye molecules bound to cellular macromolecules can be influenced by physical and chemical factors near the binding site, such as solvent polarity, cations, pH, energy transfer, excited-state reactions, and quenching. Spectroscopic methods also have been developed to record fluorescence emission spectra on each cell in real time and to resolve multiple emission spectra signals using computational methods. If fluorochromes cannot be resolved by these means, but have separated excitation spectra, multiple excitation sources can be employed to sequentially excite cells and spatially resolve the spectrally overlapping fluorescence signals.

Membrane capacitance increases induced by histamine and cyclic AMP in single gastric acid-secreting cells of the guinea pig.

During acid secretion, gastric parietal cells undergo profound morphological changes including formation of the apical secretory membrane. To examine the mechanism of histamine-induced increases in the apical membrane area at the single cell level, we monitored the membrane capacitance by applying a time-resolved phase-sensitive detection method to singly isolated parietal cells of guinea pig. A real-time increase in the membrane capacitance was detected within several min after stimulation with histamine. An H2-blocker (cimetidine), but not an H1-blocker (pyrilamine), inhibited the histamine response. Dibutyryl cyclic AMP mimicked the histamine effect. The capacitance response to histamine was sensitive to cytosolic Ca2+, temperature and N-ethylmaleimide. The histamine response was inhibited by intracellular application of a non-hydrolyzable ATP analog (AMP-PNP) and an isoquinolinesulfonamide derivative that works as an inhibitor of protein kinase A (H-8). These results indicate that in parietal cells, elevation of intracellular cyclic AMP induces exocytotic insertion of intracellular membranes into the plasma membrane, presumably by activating protein kinase A.

A novel signal processing scheme for a fluorescence based fiber-optic temperature sensor

A novel signal processing approach, which results in a significant improvement in the measurement resolution for a fluorescent decay-time-based fiber optic temperature sensor, is proposed. The technique discussed is based on the phase sensitive detection (lock-in) of the fluorescent signal. As with the other phase-sensitive detection techniques, it has the significant advantage of a high noise-suppression ability, but it differs from existing phase sensitive detection methods in several aspects which are discussed to yield this improvement. When the system is used to monitor the fluorescence lifetime of ruby, in the range from 40 °C to 100 °C, the measurement resolution achievable due to the signal processing scheme is ±0.04 °C, and from 100 °C to 200 °C, it is ±0.2 °C. It should be noted that other measurement errors must be reduced to achieve a high accuracy of measurement comparable with this. Finally, the proposed technique can be used not only for the monitoring of fluorescence lifetime, but also for the highly accurate monitoring of the time-constants of other appropriate first order systems, such as a capacitive sensor, operating under high noise conditions.

Effects of external calcium on acetylcholine-evoked increases in membrane capacitance in rat pancreatic acinar cells.

Effects of external calcium on acetylcholine-induced increases in membrane capacitance and conductance were investigated with the patch-clamp technique in combination with the phase-sensitive detection method, in single dialysed pancreatic acinar cells of rats. Both increases depended on an increase in [Ca2+]i, and a high concentration of EGTA in the cell-dialysing solution made ACh ineffective. In acinar cells exposed to a bathing solution containing the normal concentration of Ca2+ (2.5 mM CaCl2), the increase in membrane capacitance was transient and synchronous with that in membrane conductance (current) in response to 0.2 microM acetylcholine. However, in a bathing solution without CaCl2 and with EGTA (0.2 mM), the increase in membrane capacitance was sustained after the membrane conductance recovered to the original level during the ACh-stimulation. The evidence suggests that external calcium facilitates either the resealing of the fusion- or fission-pores formed at the contact between the secretory granule and the luminal cell membrane, or the membrane retrieval (endocytosis) in Ca2+-dependent exocytosis.

Agonist‐induced changes in cell membrane capacitance and conductance in dialysed pancreatic acinar cells of rats.

1. Single acinar cells enzymatically isolated from the rat pancreas were subjected to tight-seal whole-cell recordings. Changes in cell membrane capacitance and conductance were simultaneously recorded using a phase-sensitive detection method. 2. Acetylcholine (ACh, 0.05-0.5 microM) and cholecystokinin octapeptide (CCK-8, 10-50 pM) concomitantly induced transient increases in cell membrane current, capacitance and conductance only when cytosolic Ca2+ was weakly chelated by EGTA (70 microM). These responses were prolonged when the cells were dialysed with a solution containing GTP gamma S (a stable analogue of GTP, 50-100 microM), whereas they were inhibited by dialysing with that containing GDP beta S (a stable analogue of GDP). These results suggest that a type of guanine-nucleotide-binding protein (G-protein) could be involved in ACh- or CCK-receptor signalling. 3. The ACh- or CCK-induced responses (with or without GTP gamma S in the cytosol) were all abolished when a high dose of EGTA (1-2 mM) was injected into the acinar cells. In addition, A23187, a calcium ionophore, induced sustained responses when the cytosolic Ca2+ was weakly buffered by 70 microM-EGTA. These results suggest that the secretagogues regulate the changes in cell membrane capacitance and conductance via an increase and decrease of cytosolic Ca2+ concentration. 4. Oscillatory changes in cell membrane conductance and capacitance were consistently observed even without applying secretagogues when the cells were dialysed with a solution containing GTP gamma S (50-100 microM) and cytosolic free Ca2+ ions weakly buffered at about 10(-6) M with a low dose of EGTA and CaCl2. 5. The peak amplitude of changes in cell membrane capacitance induced by ACh or CCK-8, with or without GTP gamma S in the cytosol, varied between 200 and 1000 fF, thereby suggesting that 20-100 zymogen granules can fuse with the luminal cell membrane in response to these agonists in rat exocrine pancreatic acinar cells.

A patch‐clamp study of mammalian platelets and their voltage‐gated potassium current.

1. Mammalian platelets were freshly isolated from human, rabbit, or rat blood. The whole-cell and cell-attached voltage-clamp variations of the patch-clamp technique were employed to study the passive electrical properties and ion channels of unstimulated platelets. 2. The input capacitance of a platelet measured by the phase-sensitive detection method was about 128 fF, the input resistance of a platelet was about 59 G omega and the resting membrane potential was about -50 mV which was directly measured by a whole-cell recording in the current-clamp mode. 3. The predominant ion channel was a voltage-gated K+ channel resembling the delayed rectifier K+ channel of nerve, muscle and T-lymphocyte. There was no indication of any inward current in the platelet membrane. The activation of the K+ current could be fitted by n4 kinetics, and was half-maximal at about -35 mV. 4. The time constant of K+ current inactivation was virtually independent of voltage and varied from cell to cell. Recovery from inactivation was slow and dependent on the size and duration of the preceding conditional voltage step. Steady-state inactivation was half-maximal at about -50 mV and was complete at positive potentials. 5. The predominant single-K+-channel conductance was 9 pS and the estimated number of K+ channels per platelet was about 325, corresponding to a density of 25/micron2 apparent membrane area.

Ca2+-induced excess capacitance fluctuation studied by phase-sensitive detection method in exocrine pancreatic acinar cells

A phase-sensitive detection method in combination with whole-cell voltage-clamp was applied to exocrine pancreatic acinar cells from rat. The results have shown that cell-aialysis with a solution containing 5 X 10(-7) M Ca2+ induces stp-wise changes in cell capacitance. The size distribution of increasing and decreasing capacitance steps is compatible with the idea that these capacitance steps are the results of individual granular fusion and retrieval events. This method may enable experiments studying the exo- and endo-cytotic mechanisms in morphologically polarized secretory cells.

Diode-laser spectroscopy of supersonic free jets

A supersonic-free-jet infrared spectrometer has been constructed for investigation of molecular vibrational spectra at low rotational and vibrational temperatures. The sensitivity of measurement in a pulsed jet is increased by employing a phase-sensitive detection method synchronized with the pulse frequency. The performance of the spectrometer is examined for the absorption lines of the NH3v2 band. A rotational temperature as low as 16K is attained when seeded in He. Cold-jet spectra are demonstrated for thev3 bands of PF5,34SF6, and182WF6.

Direct recording of the initially excited and the solvent relaxed fluorescence emission spectra of tryptophan by phase sensitive detection of fluorescence.

Abstract Phase sensitive detection of fluorescence was used to directly record the initially excited and the solvent‐relaxed emission spectra of N‐acetyl‐L‐tryptophanamide in propylene glycol. Emission from the initially excited state was suppressed by adjusting the phase sensitive detector to be out of phase with the emission on the short wavelength side of the fluorescence spectrum. Then, the phase sensitive intensities revealed the emission spectrum of the solvent relaxed state. Similarly, the emission from the solvent relaxed state was suppressed by adjusting the detector to be out of phase with the emission on the long wavelength side of the spectrum, allowing the spectrum of the initially excited state to be directly recorded. Distinct emission spectra could be recorded when the solvent relaxation time was comparable to the fluorescence lifetime. At higher or lower temperatures, emission occurs predominantly from a single state, and suppression of the fluorescence signal at any arbitrary wavelength resulted in suppression of the entire emission. A simple theory is described which allows the spectral relaxation times to be estimated from the phase sensitive intensities. From this analysis we obtained an activation energy for spectral relaxation of 3 kcal/mol. This activation energy is smaller than that found for the temperature dependence of fluorescence depolarization, 7.8 kcal/mol. We attribute this difference to the smaller molecular motions required for spectral relaxation.The method of phase sensitive detection of fluorescence shows excellent resolving power and sensitivity, and this method should facilitate measurement of spectral relaxation around tryptophan residues in proteins.

Studies of phase characteristics of various photon echoes with a phase sensitive detection method

Various optical coherent transient signals in ruby have been examined by using an optical phase sensitive detection (PSD) technique. The PSD is performed by comparing the phase of the heterodyne beat signals obtained by the Stark switching technique with that of a local oscillator whose frequency is equal to the heterodyne beat frequency. The optical FID, two-pulse photon echoes, radiation locked echoes and notched echoes have been detected as υ-mode signals with small u-mode signals probably due to the asymmetry of the inhomogeneous broadening. The dependence of the phase of the radiation locked and notched echoes upon the driving pulses is also examined. The experimental results are in good agreement with theoretical predictions based on Bloch equations.

Phase-sensitive detection of electron-beam-induced desorption from ZnO

We report a method of phase-sensitive detection of desorption from surfaces bombarded by a properly modulated electron beam. Mass-spectrometer signals detected by this method assure a high sensitivity and selectivity of the results. This method is used here for studying surfaces of ZnO powders, and it found that CO2 is the only desorbed species during electron bombardment of such surfaces. Following the CO2 desorption kinetics simultaneously with the associated surface conductivity change, we find one-to-one correspondence between both quantitites. Furthermore, the rates of the electron-induced CO2 desportion and the surface conductivity are in a very good agreement with a theoretical model, previously developed for photodesorption. This model predicts a slowing of the desorption rate due to the buildup of an electron accumulation layer and a corresponding band bending which hinder irradiation-generated holes from reaching the surface and neutralizing CO−2 species chemisorbed on it. Our observations seem to support this model together with pointing out the similarities between electron- and photon-induced desorption in this case.

Critical magnetic studies of Tb by AC techniques

ferromagnktiques. Abstract. - The experimental results of an AC investigation into time-dependent magnetization effects in Tb over the range 77-300 K using AC susceptibility x (2-400 A m-I), transient enhancement of x and thermal modu- lation techniques are described. Our results confirm the existence of magnetic viscosity below T, (- 220 K) and we also observe thermal relaxation processes in this region. Thermal hysteresis and the transiently enhanced x in the restricted anti-ferromagnetic region (220-229 K) provide evidence for the presence of anti-ferromagnetic domains. 1. Introduction. - Previous magnetization investi- gations of Tb have shown the presence of time depen- dent and thermal hysteresis effects in the ferromagnetic region. However these studies (I-31, while concurring in their overall behaviour, show several inconsistencies in the quantitative description of the effects. Belov et al. (I) reported a magnetic viscosity for polycrys- talline Tb (H - 4 000 A m- ') below T, with rela- xation times - 100 s, whereas del Moral and Lee (2), in a single crystal'Study of AC x (H - 40 A m-', f - 21 HZ), observed a similar effect but only over the temperature range 135-205 K and with relaxation times - 1-2 h. Earlier, Jelinek et al. (3) (H - 800 A m- l) had failed to observe any magnetic viscosity but reported a 4 K temperature hysteresis in AC x around T, for polycrystalline Tb. This was in contrast with (2) who reported no apparent tem- perature hysteresis at T,. An aim of the present AC experiments has therefore been to re-examine and extend the study of the magnetic time-dependent and thermally activated processes in Tb. 2. Results and discussion. - The sample assembly was similar to that described previously (4) and experimental measurements of AC X, transient enhan- cement and thermal modulation (4, 51 were carried out in a conventional liquid nitrogen exchange gas cryostat using phase sensitive detection methods. The toroidal sample (id 12.3 mm, od 14.7 mm) is 99.9 wt % pure.

The modified Beer—Lambert law and the curve of growth for the atomic resonance transition Sn(6s( 3P o1) ← 5p 2( 3P 0))

We present a detailed study of the curve of growth for the resonance transition of ground state atomic tin Sn(6s( 3P o 1) → 5p 2( 3P 0)) at λ = 286.3 nm. An experimental curve of growth is reported from resonance absorption measurements for the λ = 286.3 nm transition on atomic tin vapour in equilibrium with solid tin in the temperature range T = 943–1238 K obtained using the method of phase sensitive detection. Expressing the extent of resonance absorption by the modified Beer—Lambert law ▪ or ▪ the logarithmic form of the experimental curve of growth yields γ = 0.65 ± 0.02. This is found to be in good agreement with previously determined empirical calibrations for this transition derived from time-resolved resonance absorption measurements (γ = 0.64 ± 0.09 and γ = 0.66 ± 0.03). Curve of growth calculations, including the effects of nuclear hyperfine interaction, using the three-layer model are found to be unable to generate departures of this magnitude from the standard Beer-Lambert law employing sensible parameters in the model. This is entirely in accord with previous results of similar measurements on the resonance transition Pb(7s( 3P o 1) ← 6p 2( 3P 0)) at λ = 283.3 nm.

The pyroelectric signal from triglycine sulphate

The pyroelectric signal from triglycine sulphate has been studied by a focussed He-Ne laser beam and a phase sensitive detection method. It is observed that anti-parallel domains give rise to pyroelectric signals of opposite polarity. The pyroelectric signal is zero when the laser spot is in between anti-parallel domains. The intensity of the pyroelectric signal is recorded and the domain structure is determined.

Nature of the broadband luminescence center in MgO:Cr3+

The luminescence spectrum of MgO:${\mathrm{Cr}}^{3+}$ is characterized by a series of sharp features at around 7000 \AA{} and a broad band which stretches from about 7000 \AA{} to 1 \ensuremath{\mu}m. The nature of the centers responsible for this broadband luminescence is examined by a combination of phase-sensitive detection methods and polarized absorption and luminescence methods. The phase-sensitive detection method allows us to separate clearly the broadband luminescence from the remainder of the spectrum and a sharp no-phonon line associated with the broadband luminescence is uncovered. The centers responsible for the broadband luminescence are found to have axes of symmetry along [011]-type directions, supporting the conclusion that this luminescence originates on ${\mathrm{Cr}}^{3+}$ ions in rhombic centers. At high temperatures broad emission is also observed from cubic site ${\mathrm{Cr}}^{3+}$ ions. The cubic site luminescence is shown to behave in accordance with theoretical expectations.

Raman difference spectra of solutions of electrolytes in formamide

A phase-sensitive detection method for recording Raman difference spectra is described. The method has been applied to concentrated solutions of LiNO3, LiCl, and NaNO3 in formamide in order to determine the separate effects of anion and cation changes on the solvent spectrum. The Raman data are interpreted in terms of the ion solvation effects, and interionic association is demonstrated.

A multichannel phase-sensitive detection method using orthogonal square waveforms

A system has been developed for the analysis of signals that vary in a linear manner with several parameters. The method allows simultaneous extraction of those parts of the signal which vary as each modulation parameter, and also any parts which depend on chosen products of the modulation parameters. The method has been used to measure small shifts of resonance frequency in molecular beam experiments, when a sensitivity approaching the limit set by shot noise has been achieved.

Nuclear Quadrupole Resonance by Nuclear Induction: Theory and Experiment; with an Extension of the Theory to Absorption Methods

A theory is presented for nuclear quadrupole resonance (NQR) by means of nuclear induction and is extended to absorption methods. Based on the Bloch-type equations of Bloom, Robinson, and Volkoff, the theory is derived for the case $I=1$, $\ensuremath{\eta}=0$. For the induction method to work, a small magnetic field must remove the degeneracy of the $m=\ifmmode\pm\else\textpm\fi{}1$ levels; in practice this is done by an audio-frequency modulation field. Phase-sensitive detection methods are then used to minimize noise. The theory predicts the effect of modulation field, static magnetic field, and rf field on the slow-passage induction signal. Theory and experiment agree that magnetic fields (modulating or static) broaden the line, but for low enough fields, the true NQR derivative line is observed. Sufficiently large static fields introduce complex structure into the line shape. The effect of rf is given by a modified form of the Bloembergen-Purcell-Pound saturation curves. Experiments on polycrystal hexamethylenetetramine yield good agreement with all aspects of the theory. The extension of the theory to single-coil (absorption) NQR methods predicts that sinusoidal magnetic modulation and phase-sensitive detection will produce zero signal in a single-coil experiment. In addition it yields the proper line shapes for Zeeman modulation and frequency modulation methods and predicts the effects of modulation amplitude and magnetic fields in both cases. A brief qualitative discussion of cases other than $I=1$, $\ensuremath{\eta}=0$ indicates that the induction method can be used for half-integral spins with any $\ensuremath{\eta}$, but that it is applicable to integral spins only when $\ensuremath{\eta}$ is small. In other words the induction method can be used for any case to which the Zeeman-modulated absorption method is applicable.