Time-dependent Intensity Changes Research Articles

Confocal Raman Microscopy Investigation of Self-Assembly of Hybrid Phospholipid Bilayers within Individual Porous Silica Chromatographic Particles.

Hybrid-supported phospholipid bilayers are a model structure utilized for measurement of molecular interactions that typically occur at cell membranes. These membrane models are prepared by adsorption of a lipid monolayer onto a stable n-alkyl chain layer that is covalently bound to a support surface. Hybrid bilayers have been adapted to chromatographic retention measurements of lipophilicity through the assembly of a phospholipid monolayer onto n-alkane-modified silica surfaces in reversed-phase chromatographic particles. Recent Raman microscopy studies of these particles have shown that the acyl chains of the phospholipid interact with the C18-alkyl chains immobilized on the silica surface, where both lipid and C18 alkyl chains become ordered because of chain interdigitation. Confocal Raman microscopy has also been used to investigate the association of small molecules with hybrid-lipid bilayers in C18 chromatographic silica particles; the partitioning of model solutes compares favorably to that in lipid vesicle membranes with similar changes in acyl-chain structure (disordering) with solute partitioning. The present study seeks information about how these membrane-mimetic bilayers assemble onto the C18-derivatized silica surfaces of reversed-phase chromatographic silica particles. Confocal Raman microscopy is capable of interrogating the time-dependent internal composition and structure within individual silica particles. The Raman scattering data can be resolved into component Raman spectra and corresponding composition vectors that describe the time-dependent changes in intensity of the component spectra. This analysis provides insight into how the structures of both the lipid and C18 alkyl chains of hybrid lipid bilayers evolve during deposition and organization on the internal surfaces of reversed-phase chromatographic silica particles.

Real-Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy.

The kinase inhibitory domain of the cell cycle regulatory protein p27Kip1 (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D-DNP). While intrinsically disordered in isolation, p27 adopts secondary structural motifs, including an α-helical structure, upon binding to cyclin-dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real-time observation of 13 C NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time-dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. The analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D-DNP NMR experiment.

Photoexcitation Dynamics of NO-Bound Ferric Myoglobin Investigated by Femtosecond Vibrational Spectroscopy

Femtosecond vibrational spectroscopy was used to investigate the photoexcitation dynamics of NO-bound ferric myoglobin (Mb(III)NO) in D2O solution at 294 K after excitation with a 575 nm pulse. The stretching mode of NO in Mb(III)NO consists of a major band at 1922 cm(-1) (97.7%) and a minor band at 1902 cm(-1) (2.3%), suggesting that Mb(III)NO in room temperature solution has two conformational substates. The time-resolved spectra show small but significant new absorption features at the lower-energy side of the main band (1920-1800 cm(-1)). One new absorption feature in the region of 1920-1880 cm(-1) exhibits the (15)NO isotope shift (37 cm(-1)) the same as that of the NO band in the ground electronic state of Mb(III)NO. This absorption shifts toward higher energy and narrows with a time constant of 2.4 ps, indicating that it evolves with rapid electronic and thermal relaxation of the photoexcited Mb(III)NO without photodeligation of the NO from the heme. Absorption features assigned to proteins undergoing thermal relaxation without NO deligation add up to 14 ± 1% of the total bleach, implying that the photolysis quantum yield of Mb(III)NO with a Q-band excitation is ≤0.86 ± 0.01. The remaining absorption bands peaked near 1867, 1845, and 1815 cm(-1), each showing the (15)NO isotope shift the same as that of the free NO radical (33 cm(-1)), were assigned to the vibrational band of the photodeligated NO, the NO band of Mb(III)NO in an intermediate electronic state with low-spin Fe(III)-NO(radical) character (denoted as the R state), and the NO band of the vibrationally excited NO in the R state, respectively. A kinetics model successfully reproducing the time-dependent intensity changes of the transient bands suggests that every rebound NO forms the R state that eventually relaxes into the ground electronic state nonexponentially. Most of the photodissociated NO undergoes fast geminate recombination (GR), and the rebinding kinetics depends on the conformation of the protein. GR of NO to Mb(III) in the major conformation shows highly nonexponential kinetics described by a stretched exponential function, exp(-(t/290 ps)(0.44). The NO rebinding to Mb(III) in the minor conformation is exponential, exp(-t/1.8 ns), suggesting that the distal histidine, the interaction of which dictates the conformation of Mb(III)NO, participates in mediating the binding of NO to Mb(III). In Mb(III)NO, the elusive low-spin Fe(III)-NO(radical) state, proposed in electronic structure calculations, indeed exists at >12 kJ/mol above the ground state and takes part in the bond formation of Fe(III)-NO, suggesting that it plays a significant role in the function of NO-bound ferric protein. Time-resolved vibrational spectra with high sensitivity reveal rich photophysical and photochemical processes of photoexcited Mb(III)NO.

Contact Angle Dependence of the Resonant Frequency of Sessile Water Droplets

The resonant vibrations of small (microliter) sessile water droplets supported on solid substrates were monitored using a simple optical detection technique. A small puff of air was used to apply an impulse to the droplets and their time dependent oscillations were monitored by passing a laser beam through the droplet and measuring the variations of the intensity of the scattered light using a simple photodiode arrangement. The resulting time dependent intensity changes were then Fourier transformed to obtain information about the vibrational frequencies of the droplets. The resonant frequencies of droplets with masses in the range 0.005-0.03 g were obtained on surfaces with water contact angles ranging from 12 ± 4° to 160 ± 5°. The contact angle dependence of the resonant frequency of the droplets was found to be in good agreement with a simple theory which considers standing wave states along the meridian profile length of the droplets.

Evaluation of Escherichia coli cell response to antibiotic treatment by use of Raman spectroscopy with laser tweezers.

Laser tweezers Raman spectroscopy was used to detect the cellular response of Escherichia coli cells to penicillin G-streptomycin and cefazolin. Time-dependent intensity changes of several Raman peaks at 729, 1,245, and 1,660 cm(-1) enabled untreated cells and cells treated with the different antibiotic drugs to be distinguished.

The role of time-dependent memory processes in the development of PTSD-like symptoms in mice

The primary symptoms of Posttraumatic stress disorder (PTSD) were traditionally interpreted as an extraordinary strong memory of the trauma and hyperarousal, with the latter increasing with the passage of time (fear incubation) and evolving into generalized fear and generalized avoidance behaviour. Here we studied time-dependent changes in intensity and specificity of associative fear, as well as hyperarousal levels and avoidance behaviour in our mouse model of PTSD. Testing the animals at different time points after trauma, we observed a consistent level of conditioned fear and hyperarousal. On the other hand, context discrimination ratio decreased over time, whereas generalization of avoidance behaviour increased. Based on these time-dependent changes in the development of PTSD-like symptoms, we assumed that they might be differentially affected by early vs. late extinction training. Both early and late extinction training led to a long-lasting reduction of contextual and generalized fear, but indeed only early extinction caused an amelioration of hyperarousal and went ahead without spontaneous recovery of contextual fear. Taken together our data indicate that (i) fear incubation and generalized avoidance phenomena are not a result of an increase in hyperarousal, but a time-dependent reduction in recalling the details of trauma context, (ii) hyperarousal is largely independent of associative fear and relatively stable over time, (iii) early and late post-trauma extinction training might represent two qualitatively different processes and (iv) early post-trauma intervention is particularly important in order to prevent the development of long-lasting trauma-associated memories and hyperarousal.

In situ ATR-FTIR studies of the aluminium/polymer interface upon exposure to water and electrolyte

Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) with the Kretschmann configuration was applied for in situ studies of the transport of water and ionic species through a polymer film to an aluminium/polymer interface. The time dependent intensity changes of the infrared bands of water were used to follow the transport of water to the aluminium/polymer interfacial region and a NaSCN solution was employed as model electrolyte to follow the transport and accumulation of thiocyanate ions. Apart from water sorption and ion transport, the main processes identified were corrosion/oxidation of the aluminium surface and swelling of the polymer film. The method proved to be useful for detailed in situ studies of changes at a polymer coated metal surface, such as oxidation and surface film formation on the metal. It should also be possible to study the effects of defects and pores in the polymer film on the transport properties of water and ions to the metal/polymer interface, as well as adsorption and other chemical reactions and physical interactions in the metal/polymer interfacial region.

Direct monitoring of lipid oxidation in edible oils by Fourier transform Raman spectroscopy

Fourier transform Raman spectroscopy has been used to investigate the chemical changes taking place during lipid oxidation in several edible oils. Oxidative degradation of six vegetable oils was accelerated by heating at 160 °C. Formation of aldehydes was detected, and saturated as well as α,β-unsaturated aldehydes could be identified with the help of pure component spectra. The formation of conjugated double bond systems and the isomerisation of cis to trans double bonds was observed in the C C stretching region and found to follow a distinct pattern for the different oils. It was possible to associate these differences to the fatty acid composition. The time-dependent intensity changes in certain Raman bands were compared to conventional parameters used to determine the extent of oxidation in oils, such as anisidine value and K 270, and showed good correlation.

Postoperative magnetic resonance imaging findings after transtemporal and translabyrinthine vestibular schwannoma resection.

Magnetic resonance imaging (MRI) has become the investigation of choice to follow up patients after vestibular schwannoma resection. Retrospective. Postoperative MRI findings of 70 patients after vestibular schwannoma resection through a transtemporal (n = 48) and a translabyrinthine (n = 22) approach were reviewed. Time-dependent changes in intensity, size, and shape of enhancement in the internal auditory canal before and after contrast administration, postoperative temporal lobe gliosis, and changes of fat grafts were evaluated. After vestibular schwannoma resection, all patients showed signal enhancements in the internal auditory canal ranging from a faint to high signal intensity in the first postoperative MRI, 3 to 6 months after surgery. In the next MRI at 12 to 24 months after surgery, 30 patients (43%) showed a decreased signal, 35 patients (50%) a stable enhancement, and 5 patients (7%) an increased enhancement in the internal auditory canal depicted as an intense nodular or mass-like pattern. In patients with decreased or stable enhancement, a residual tumor could be excluded in the following MRI scans, whereas in all patients with increased enhancements after 12 to 24 months, signal enhancement further increased and residual tumors were detected. Different degrees of temporal lobe gliosis were found in 15 of 48 cases (31%) after transtemporal tumor removal. Enhancement of fat grafts used in 22 cases decreased to different degrees in 14 cases (64%). Differentiation of residual tumor from scar tissue in the internal auditory canal after vestibular schwannoma resection requires close, long-term follow-up. Nodular and progressive enhancements in the internal auditory canal indicate residual tumor. Linear enhancement in the internal auditory canal has been found to be a common finding after vestibular schwannoma resection not associated with residual tumor.

Rovibrational transitions and nuclear spin conversion of methane in parahydrogen crystals

Solid parahydrogen is an excellent matrix for matrix-isolation spectroscopy because of its high spectral resolution. Here we describe the rovibrational structure and nuclear spin conversion of CH4 embedded in parahydrogen crystals studied by infrared absorption spectroscopy. The vibration–rotation absorptions of CH4 exhibit time-dependent intensity changes at 4.8 K. These changes are interpreted to be a result of the I=1→I=2 nuclear spin conversion that accompanies the J=1→J=0 rotational relaxation. The half-lifetime of the upper J=1 rotational state is unchanged by the addition of up to 2% orthohydrogen molecules but decreases with more than 10% orthohydrogen molecules. The increase of the decay rate at higher orthohydrogen concentration indicates that the magnetic field gradient across CH4 due to the orthohydrogen molecules mixes the nuclear spin states, which accelerates the conversion.

Surface-Enhanced Raman Scattering of Biological Molecules on Metal Colloid II: Effects of Aggregation of Gold Colloid and Comparison of Effects of pH of Glycine Solutions between Gold and Silver Colloids

As the second paper of a series of our studies on surface-enhanced Raman scattering (SERS) of biological molecules on metal colloid, this paper reports effects of aggregation of gold colloid on the SERS intensity of glycine (Gly) in aqueous solutions and comparison of effects of pH of the Gly solutions between gold and silver colloids. In order to investigate the relation between the SERS effect and colloidal aggregation, we have developed a spectroscopic system by which we can measure simultaneously time-resolved SERS spectra and a time-dependent intensity change in a surface plasmon absorption of a metal colloid system. By use of this instrument we monitored time-dependent intensity changes in the SERS signals and in an absorption band at 802 nm of Gly on gold colloid at pH 3.9 and 6.0. For both pH values, the intensities of the SERS and the absorption band change in concert with each other at the emergence of the SERS effect. The intensity changes in the SERS signals are rather slow for the solution of pH 6.0 while rapid for that of pH 3.9. The coagulation proceeds slowly near the isoelectric point of Gly, whereas the rapid coagulation happens in the acidic region. SERS spectra of Gly adsorbed on silver colloid were measured at various pH values, and it was found that, in sharp contrast to the gold colloid system, which we reported in our previous paper, the SERS intensity of Gly on the silver colloid decreases markedly at pH 2.0 where most of Gly in the solution has a COOH group instead of a COO− group. It seems that the coagulation of the silver colloid particles does not occur very much below p K1 (2.35) because the COOH groups do not interact strongly with the positively charged silver particles.

Kinetics of radiation- and cytochrome c-induced modifications in liposomes analysed by FT–Raman spectroscopy

Fourier transform Raman spectroscopy on artificial lipid membranes was used to study radiation-induced peroxidation processes as a function of time after radiation exposure. The time dependent intensity changes of the Raman lines of various CC bondings were compared to results obtained by measuring conjugated dienes and by the thiobarbituric acid test for malondialdehydes. The results show that mainly the cis CC bonds of the lipid chains are involved and, therefore, indicate that γ-radiation induces conformational changes in the lipid chain while the mobility of the lipid chains is reduced. New Raman bands can be assigned to aldehyde products induced at the end of the peroxidation process. The immediate decrease of the CH vibration lines was directly correlated with the formation of conjugated CC double bonds suggesting that these vibration lines are in contrast to the CC lines solely Raman active, when isolated CC bonds are present. Cytochrome c (ox.) incorporated into the bilayer of the artificial membranes induced autooxidation processes not influenced by γ-radiation. It was observed that cytochrome c (ox.)-induced changes of the relative intensity of the CC bonds differ from those induced by γ-radiation. These results of cytochrome c together with the inhibitory effects of the antioxidant α-tocopherol suggest that the radical species involved in the cytochrome c induced process might be different from the free radicals involved in the γ-radiation-induced process.

Asynchronous Time-Resolved FT-IR Study of the Dynamical Behavior of Ferroelectric Liquid Crystal with a Tolane Ring

Transient infrared spectra of a ferroelectric liquid crystal with a tolane ring, ( S)-4-methylhexyl-4-[4-(decyloxy)phenylethynyl]-2-fluorobenzate, in the smectic C phase have been measured under various temperatures and voltages by use of an asynchronous time-resolved FT-IR method. The effects of temperature and voltage on the rate and tilt angle of the electric field-induced reorientation of the molecule have been studied. The absolute values of the observed intensity changes and their sign during the switching can be explained by the static properties of the sample. The spectra obtained under the different experimental conditions suggest that the temperature and applied voltage alter the tilt angle and angular velocity of reorientation of the liquid crystal, respectively. Dominant infrared bands show very similar time-dependent intensity changes under various conditions, indicating that the whole molecule reorients simultaneously irrespective of temperature and the applied field strength as though the molecule were a rigid rod.

Separability of Intra- and Intermolecular Vibrational Relaxation Processes in the S1 State of trans-Stilbene

Detailed time-resolved measurements of anti-Stokes intensities of several high-frequency modes of electronically excited trans-stilbene in hexane and methanol are reported. The most striking changes are observed in the ethylenic band at 1570 cm-1. Nonequilibrium population in the v = 1 level of this mode is found to exhibit a biphasic decay, which is interpreted as a rapid energy redistribution followed by a slightly slower process that reflects the transfer of excess energy to the solvent. Comparison of the time-dependent intensity changes of several high-frequency bands with those from a model that assumes instantaneous thermalization of the excited state suggests that internal energy randomization is incomplete on the time scale of solute−solvent thermal equilibration process. No evidence of mode specificity in the energy redistribution process is observed. The results show us to place a value of ∼2 ps on the lifetime of the v = 1 level of the ethylenic stretching mode of trans-stilbene in both hexane and methanol. Conclusions from this work are discussed in the context of other recent time-resolved Raman measurements of vibrational relaxation in this system in both the gas phase and condensed phase.

Protein Dynamics in the Bacteriorhodopsin Photocycle: A Nanosecond Step-Scan FTIR Investigation of the KL to L Transition

A time-resolved step-scan FTIR spectrometer with a time resolution of 20 ns was developed and used to investigate the KL to L transition in the photocycle of bacteriorhodopsin in the time range from −60 to 940 ns. Broadband FTIR absorbance difference spectra with a spectral range of 850−2050 cm-1 and a spectral resolution of 4 cm-1 have been obtained. Our data show that there are two sets of BR photoproduct difference bands exhibiting different kinetics. The intensity changes of bands attributed to structural changes of the carboxyl groups Asp-96 and Asp-115 as well as of bands assigned to CC and CC stretching modes of the chromophore show single exponential behavior with a time constant of about 400 ns, implying that these two processes are coupled. The time-dependent intensity changes of bands attributed to structural changes of the chromophore region near the Schiff base exhibit slower kinetics with a time constant of about 2 μs. We interpret our data in terms of a process during the KL to L transition where structural changes of the β-ionone ring end of the chromophore and of Asp-115 occur faster than changes at the Schiff base region of the chromophore. Under our physiological sample conditions, a perturbation of Asp-115 occurs in the first 20 ns in contrast to results from hydrated films where this process is blocked or occurs more slowly. This fast protein response indicates that there is direct coupling between the carboxylic acid residues and the chromophore. Comparison of our data with low-temperature and microsecond time-resolved infrared spectra of the L intermediate in hydrated films of BR indicates that a different L structure is produced when the water activity is low.

Characterization of the growth of sol-gel derived BaTiO thin films by laser Raman spectroscopy

The isothermal growth kinetics of sol-gel derived BaTi 5O 11 thin films has been studied by laser Raman spectroscopy. Thin films of the precursors were spin coated on silicon wafers with platinum or gold overlayers. The films were then annealed isothermally, and Raman spectra were recorded in situ. The time-dependent intensity changes of the Raman bands could be analyzed in terms of Avrami models. The interaction of the BaTi 5O 11 film with the platinum or gold overlayers during heat treatment apparently increased the Raman intensity by a factor of about 100.

Theory of Time-resolved Resonance Raman Scattering from Vibrationally Hot Molecules

Abstract A theory of time-resolved resonance Raman scattering (RRS) from molecules is developed. The time-resolved RRS cross section is defined as a product of the time-dependent vibronic energy distribution and the scattering cross section from the single vibronic level. The theory is applied to the time-resolved RRS from vibrationally hot molecules whose vibronic distribution characterized by a canonical distribution with temperature higher than that of the heat bath relaxes to the equilibrium one in the presence of a collisional vibrational relaxation. Analytical expressions for the time-resolved RRS cross section are derived within a displaced harmonic oscillator model. It is shown that in the strong coupling case the intensities of the Stokes and anti-Stokes Raman bands in the rigorous resonance case vary inversely as the time-dependent average vibrational occupation number. Some model calculations are performed to illustrate the time-dependent intensity changes of the Raman bands.