Breaking the nanosecond barrier in FTIR time-resolved spectroscopy

The state of the art in broad spectral bandwidth infrared time-resolved spectroscopy (IRTRS) is reviewed, with particular regard to time resolution in the nanosecond and sub-nanosecond regime. While step-scan Fourier transform infrared (S²FTIR) has been successful in pushing the time resolution of IRTRS to sub-microsecond limits, and is, in principle, applicable for monitoring time-dependent phenomena on any time scale, a practical limit for S²FTIR is currently about 1 ns, due to the limitations of parts of the instrument other than the interferometer itself. For the particular case of IRTRS of transient photo-excited states illustrated here and other photo-excitation studies, it is proposed that the most effective way to breach the nanosecond barrier and to push the time resolution limit of IRTRS to 10 ps, or even lower, while still maintaining the spectral bandwidth and resolution and the multiplex and throughput advantages of interferometry, is to turn to constant velocity, continuous-scan (CS) FTIR, in the pump–probe asynchronous sampling mode. In the method described, the pump is provided by the picosecond UV pulse of an electron storage ring-powered free electron laser and the infrared probe is the picosecond `white light' synchrotron pulse from the same storage ring. The design specifications of this system are 10 ps time resolution with 3 cm⁻¹ spectral resolution.     

Set-up for time-resolved step-scan FTIR spectroscopy of noncyclic reactions

We have established a novel technique, which allows the application of time-resolved step-scan FTIR difference spectroscopy on noncyclic reactions. Cyclic reactions are ideally suited for the step-scan technique. However, it is difficult to apply the step-scan technique to noncyclic reactions, because the investigated process has to be repeated at about 1000 sampling positions of the interferogram. Consequently, to investigate noncyclic systems the sample has to be renewed at every sampling position. In the presented novel approach the IR-beam and the excitation laser-beam are focused to a very small diameter of 200 μm. Thereby, only a small segment of the sample, which has an overall diameter of 15 mm, is excited and probed. By moving the sample, which is mounted on an x-y-stage, to different nonexcited segments the reaction can be repeated until a complete interferogram data set is recorded. In so far as the typically used flow cells are concerned their optical pathlength is too large to perform difference spectroscopy. We use 4 μm thin films to depress the water background absorption of biological samples. As test, the well known photo-cyclic reactions of bacteriorhodopsin are measured. No systematic errors appear in the difference spectra. Because of intensity loss by the IR-microscope the signal-to-noise ratio is about 5 times less as compared to conventional step-scan measurements. For the first time, the technique is then applied to a noncyclic reaction, the photolysis of caged ATP. The successful performance with 10 μs time-resolution now opens the door for many new applications of step-scan FTIR measurements to noncyclic reactions.     

Proteins in action monitored by time-resolved FTIR spectroscopy.

In the post genome era proteins coming into the focus of life sciences. X-ray structure analysis and NMR spectroscopy are established methods to determine the geometry of proteins. In order to determine the molecular reaction mechanism of proteins, time-resolved FTIR (trFTIR) difference spectroscopy emerges as a valuable tool. In this Minireview we describe the trFTIR difference spectroscopy and show its application on the light-driven proton pump bacteriorhodopsin (bR), the photosynthetic reaction center and the GTPase Ras, which is crucial in signal transduction. The main principles of the technique are presented, including a summary of triggering techniques, scan modes and analysis.     

Step-scan FTIR time-resolved spectroscopy study of excited-state dipole orientation in soluble metallopolymers

Step-scan FTIR time-resolved spectroscopy (S2FTIR TRS) in acetonitrile-d3 has been used to probe the acceptor ligand in metal-to-ligand charge transfer (MLCT) excited states of amide-substituted polypyridyl complexes of RuII and in analogues appended to polystyrene. On the basis of ground-to-excited state shifts in v(C = O) of -31 cm-1 for the amide group in [RuII(bpy)2(bpyCONHEt')]2+ (bpyCONHEt' = 4'-methyl-2,2'-bipyridine-4-carboxamide-Et'; Et' = -CH2CH2BzCH2CH3) (1) and in the derivatized polystyrene abbreviated [PS-[CH2-CH2NHCObpy-RuII(bpy)2]20]40+ (3), the excited-state dipole is directed toward the amide-containing pyridyl group in the polymer side chain. Smaller shifts in v(C = O) of -17 cm-1 in [RuII(4,4'-(CONEt2)2bpy)2-(bpyCONHEt')]2+ (2) and in the derivatized polystyrene abbreviated [PS-[CH2CH2NHCObpy-RuII(4,4'-(CONEt2)2bpy)2]20]40+ (4) indicate that the excited-state dipole is directed toward one of the diamide bpy ligands. The nearly identical results for 1 and 3 and for 2 and 4 show that the molecular and electronic structures of the monomer excited states are largely retained in the polymer samples. These conclusions about dipole orientation in the polymers are potentially of importance in understanding intrastrand energy transfer dynamics. The excited-state dipole in 3 is oriented in the direction of the covalent link to the polymer backbone, and toward nearest neighbors. In 4, it is oriented away from the backbone.     

Rotational Raman spectroscopy of ethylene using a femtosecond time-resolved pump-probe technique

Femtosecond Raman-induced polarization spectroscopy (RIPS) was conducted at low pressure (250 mb at 295 K and 400 mb at 373 K) in ethylene. The temporal signal, resulting from the beating between pure rotational coherences, was measured with a heterodyne detection. The temporal traces were converted to the frequency domain using a Fourier transformation and then analyzed thanks to the D2hTDS software (http://www.u-bourgogne.fr/LPUB/shTDS.html) dedicated to X2Y4 molecules with D2h symmetry. The effective Hamiltonian was expanded up to order 2, allowing the determination of five parameters with an rms of 0.017 cm(-1). Special care was taken in the precise modeling of intensities, taking into account all instrumental effects. Relative intensities were fitted (with an rms of 7.2%) and two polarizability operators were determined.     

Surface and depth-profile analysis using FTIR spectroscopy

Summary The introduction of Fourier Transform techniques and the increasing use of computers in infrared spectroscopy has made new techniques of investigation available to the spectroscopist, such as photoacoustic spectroscopy (PAS) and IR microscopy. These methods now complement the techniques of specular reflectance and attenuated total reflectance. Thin films on metals, sometimes with a thickness of much less than a micron, can be studied by various specular reflectance methods. The physical basis of the attenuated total reflectance technique (ATR) leads to a penetration of the radiation in the order of a few microns. It is, therefore, especially suitable for the investigation of surfaces and of layers close to the surface. By changing the modulation frequency of the IR radiation, i.e. the mirror velocity of the FTIR spectrometer, photoacoustic spectroscopy (PAS) can be employed to study layers at various depths below the surface of a sample. Therefore, this technique allows depth-profile analysis, so that PAS reveals itself as a complementary method to attenuated total reflectance spectroscopy. Samples with inhomogeneous profiles, e.g. laminated polymer films, can often be prepared as microtome slices perpendicular to the layered structure. Using infrared microscopy it is now possible to investigate different regions of the cross-section easily. The size of the regions that can be studied in this way may be as small as a few microns.

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Oberflächen- und Tiefenprofilanalyse mit Hilfe der FTIR-Spektroskopie

     

Pretilt angle measurements on smectic A cells with chevron and tilted bookshelf layer structures

We have measured the pretilt angle induced by rubbed polymer films in a smectic A and in a nematic liquid crystalline medium using an optical phase retardation method. The pretilt angle was found to depend on the liquid crystalline phase (smectic A versus nematic) and on the smectic layer structure (chevron versus tilted-bookshelf). The occurrence of the different smectic layer structures was verified by X-ray diffraction measurements. The effect of the applied rubbing energy on the pretilt angle obtained is measured.     

A search for carbene-solvent interactions using time-resolved infrared spectroscopy

[reaction: see text] The time-resolved infrared (TRIR) spectra of chlorophenylcarbene (CPC) and fluorophenylcarbene (FPC) were recorded in heptane at ambient temperature. The C[bond]C and C[bond]F vibrational frequencies involving the carbene carbon were obtained in heptane, benzene, and acetonitrile and in heptane containing 0.1M tetrahydrofuran or benzene. It is concluded that carbene-solvent interactions of CPC and FPC are quite weak.     

Deconvoluting binding sites in amyloid nanofibrils using time-resolved spectroscopy

Steady-state fluorescence spectroscopy has a central role not only for sensing applications, but also in biophysics and imaging. Light switching probes, such as ruthenium dipyridophenazine complexes, have been used to study complex systems such as DNA, RNA, and amyloid fibrils. Nonetheless, steady-state spectroscopy is limited in the kind of information it can provide. In this paper, we use time-resolved spectroscopy for studying binding interactions between amyloid-β fibrillar structures and photoluminescent ligands. Using time-resolved spectroscopy, we demonstrate that ruthenium complexes with a pyrazino phenanthroline derivative can bind to two distinct binding sites on the surface of fibrillar amyloid-β, in contrast with previous studies using steady-state photoluminescence spectroscopy, which only identified one binding site for similar compounds. The second elusive binding site is revealed when deconvoluting the signals from the time-resolved decay traces, allowing the determination of dissociation constants of 3 and 2.2 μM. Molecular dynamic simulations agree with two binding sites on the surface of amyloid-β fibrils. Time-resolved spectroscopy was also used to monitor the aggregation of amyloid-β in real-time. In addition, we show that common polypyridine complexes can bind to amyloid-β also at two different binding sites. Information on how molecules bind to amyloid proteins is important to understand their toxicity and to design potential drugs that bind and quench their deleterious effects. The additional information contained in time-resolved spectroscopy provides a powerful tool not only for studying excited state dynamics but also for sensing and revealing important information about the system including hidden binding sites.

Deconvolution of binding equilibrium data measured by time-resolved spectroscopy revealed two binding sites of a ruthenium complex when bound to amyloid-β fibrils: one elongated the lifetime of ruthenium complex and the other did not affect its lifetime.     

Demonstration of cavity enhanced FTIR spectroscopy using a femtosecond laser absorption source

A proof of principle experiment was performed by recording the cavity enhanced absorption spectrum of the weak b–X transition of molecular oxygen in the atmosphere using a Ti:Sa femtosecond laser as an absorption source and a high resolution continuous scan Fourier transform interferometer. The cavity was mode matched and either continuously scanned or stabilized at the so-called magic point. An optimal rms noise equivalent absorption of 3 × 10^?7 cm^?1 Hz^?1/2 was reached in the latter case, corresponding to α_min = 3 × 10^?7 cm^?1.     

Picosecond time-resolved multiplex CARS spectroscopy using optical Kerr gating

We have developed a new spectroscopic system for picosecond time-resolved coherent anti-Stokes Raman scattering (CARS) measurements. Using the optical Kerr gating method in conjunction with a nanosecond laser-based CARS system, a time resolution of 1 ps has been achieved. All-trans retinal in 1-butanol has been measured. The observed time-resolved CARS spectra show changes in the 0–10 ps time range, which are ascribed to the photoisomerization dynamics of all-trans retinal in solution.     

Dynamics of nematic point defects in a capillary with tilted boundary conditions

The motion of a single point defect in a cylindrical cavity filled with a nematic liquid crystal is described by solving numerically the simplified equations of nematodynamics. Perfect homeotropic anchoring for the director on the lateral boundary would result in the creation of domains with equal elastic energy, escaped upwards or downwards along the cavity axis and separated by point defects of strength ± 1. Defects do not move as long as they are sufficiently far apart. However, small deviations from homeotropic anchoring remove this degeneracy and the energetically favourable domains start to expand at the expense of the others, thus setting the defects in motion along the tube. We present a new numerical approach, which neglects the backflow, for studying the influence of both the pretilt and the elastic anisotropy (K ₃₃ ≠ K ₁₁) on the motion of a defect. We show how even very small pretilt angles (≈1°) result in speeds observed in experiments. For a moderate elastic anisotropy, the velocity of a +1 defect equals the velocity of a -1 defect, whereas for K ₃₃?K ₁₁ a + 1 defect moves faster than a -1 defect. For small pretilts we confirm a good qualitative agreement with an existing analytical approach, which proves inaccurate for large pretilts.     

Determination of the molecular structure of diacetylene from high-resolution FTIR spectroscopy

The high-resolution infrared absorption spectra of eight²H or¹³C substituted isotopomers of diacetylene have been recorded, and the bands corresponding to thev ₄ fundamental andv ₆ combination of the major isotopomer have been analyzed using a Loomis-Wood-type program. Effective ground-state rotational constants have been obtained from ground-state combination differences. A number ofr ₀,r _s,r _m , and (r _m )_corr structures have been calculated from the available data and are compared to those obtained by ab initio methods. The (r _m )_corr structure, which is a reliable near-equilibrium structure of diacetylene, isr _C–H=106.131(13) pm;r _C–C=137.081(16) pm;r _C-C=120.964(14). (r _m )_corr structures of the related molecules cyanogen, cyanoacetylene, and cyanodiacetylene have been calculated, and near-equilibrium structures of triacetylene and dicyanoacetylene have been predicted.     

Discrimination between single Escherichia coli cells using time-resolved confocal spectroscopy

We describe a technique for rapidly discriminating between single-cell populations within a flowing microfluidic stream. Single-cell time-correlated single-photon counting (scTCSPC) as well as photon burst spectroscopy are used to characterize individual Escherichia coli cells expressed with either green, cyano, or yellow fluorescent protein. The approach utilizes standard confocal fluorescence microscopy incorporating femtoliter detection volumes. The measured burst width characteristics are predominately governed by the fluorescence quantum yield and absorption cross section of the proteins used. It is these characteristics which were used to distinguish between cells with high precision. By utilizing scTCSPC individual fluorescence lifetimes originating from single cells could also be determined. Average fluorescence lifetimes are determined using standard deconvolution procedures. The simplicity of the approach for obtaining well-defined burst width distributions is expected to be extremely valuable for single-cell sorting experiments.     

Space and time-resolved laser-induced breakdown spectroscopy using charge-coupled device detection

Space and time-resolved studies of laser induced plasmas in air at atmospheric pressure are presented. Photovoltaic solar cells have been used as samples. The second harmonic (532 nm) of a Nd : YAG laser at an irradiance of 18 × 10¹² W/cm² has been used. The precise focus of the beam allows a microanalysis at a 0.02 mm² surface area working in single-shot mode. The use of an intensified charge-coupled device (CCD) detector has allowed time-resolved studies in both imaging or spectroscopy modes. The two-dimensional capability of the CCD has enabled the study of atomic and ionic species distribution along the plume. Most data have been recorded using single-laser shot experiments. Spectral lines have been assigned to transitions in atomic components of the material under investigation in the neutral or ionic states of the corresponding atoms. Effects of delay in improving spectral resolution and some examples of spectral characterization of species as a function of its decay are shown.     

FTIR spectroscopy of monolayers and ultrathin films using polarization modulation

Polarization modulated IRRAS is a new differential method of Fourier transform infrared spectroscopy, used to investigate ultrathin films. Spectra of a single L.B. monolayer of cadmium arachidate and of a 100 Å thick polymer film are presented in order to illustrate the main advantages of this method: high surface sensitivity, perfect environmental signal rejection and easy to get conformational information on surface molecules.     

Space and time-resolved laser-induced breakdown spectroscopy using charge-coupled device detection

Space and time-resolved studies of laser induced plasmas in air at atmospheric pressure are presented. Photovoltaic solar cells have been used as samples. The second harmonic (532 nm) of a Nd : YAG laser at an irradiance of 18 x 10(12) W/cm(2) has been used. The precise focus of the beam allows a microanalysis at a 0.02 mm(2) surface area working in single-shot mode. The use of an intensified charge-coupled device (CCD) detector has allowed time-resolved studies in both imaging or spectroscopy modes. The two-dimensional capability of the CCD has enabled the study of atomic and ionic species distribution along the plume. Most data have been recorded using single-laser shot experiments. Spectral lines have been assigned to transitions in atomic components of the material under investigation in the neutral or ionic states of the corresponding atoms. Effects of delay in improving spectral resolution and some examples of spectral characterization of species as a function of its decay are shown.     

Dynamics of CO in mesoporous silica monitored by time-resolved step-scan and rapid-scan FT-IR spectroscopy

Carbon monoxide molecules generated in the channels of mesoporous MCM-41 silica sieve from a precursor (diphenyl cyclopropenone) by photodissociation with a nanosecond laser pulse were monitored by time-resolved Fourier transform infrared (FTIR) spectroscopy using the step-scan and rapid-scan methods. A very broad absorption of CO is observed in the region 2200-2080 cm(-1) at room temperature that decays in a biphasic mode. Two-thirds of the band intensity decays on the hundreds of microsecond scale (lifetime 344 +/- 70 micros). The process represents the escape of the molecules through the mesopores into the surrounding gas phase, and a diffusion constant of 1.5 x 10(-9) m(2)/s is derived (assuming control by intra-MCM-41 particle diffusion). The broad profile of the absorption is attributed to contact of the random hopping CO with siloxane and silanol groups of the pore surface. Measurements using MCM-41 with the silanols partially capped by trimethyl silyl groups gave further insight into the nature of the IR band profile. These are the first observations on the diffusion behavior of carbon monoxide in a mesoporous material at room temperature. The residual carbon monoxide remains much longer in the pores and features distinct peaks at 2167 and 2105 cm(-1) characteristic for CO adsorbed on SiOH groups C end on and O end on, respectively. The bands decrease with time constants of 113 +/- 3 ms (2167 cm(-1)) and 155 +/- 15 ms (2105 cm(-1)) suggesting that CO in these sites is additionally trapped by surrounding diphenyl acetylene coproduct and/or precursor molecules.     

Quantitation of the global secondary structure of globular proteins by FTIR spectroscopy: Comparison with X-ray crystallographic structure

Fourier transform infrared spectroscopy (FTIR) is potentially a powerful tool for determining the global secondary structure of proteins in solution, providing the spectra are analyzed using a statistically and theoretically justified methodology. We have performed FTIR experiments on 14 globular proteins and two synthetic polypeptides whose X-ray crystal structures are known to exhibit varying types and amounts of secondary structures. Calculation of the component structural elements of the vibrational bands was accomplished using nonlinear regression analysis, by fitting both the amide I and amide II bands of the Fourier self-deconvoluted spectra, the second-derivative spectra, and the original spectra. The methodology was theoretically justified by comparing (via nonlinear regression analysis) the global secondary structure determined after deconvolving into component bands the vibrational amide I envelopes with the calculated structure determined by first principles from Ramachandran analysis of the X-ray crystallographic structure of 14 proteins from the Brookhaven protein data bank. Justification of the nonlinear regression analysis model with respect to experimental and instrumental considerations was achieved by the decomposition of all the bands of benzene and an aqueous solution of ammonium acetate into component bands while floating the Gaussian/Lorentzian character of the line shapes. The results for benzene yield all pure Lorentzian line shapes with no Gaussian character while the ammonium acetate spectra yielded all Gaussian line shapes with no Lorentzian character. In addition, all-protein spectra yielded pure Gaussian line shapes with no Lorentzian character. Finally, the model was statistically justified by recognizing random deviation patterns in the regression analysis from all fits and by the extra sum of squares F-test which uses the degrees of freedom and the root mean square values as a tool to determine the optimum number of component bands required for the nonlinear regression analysis. Results from this study demonstrate that the globular secondary structure calculated from the amide I envelope for these 14 proteins from FTIR is in excellent agreement with the values calculated from the X-ray crystallographic data using three-dimensional Ramachandran analysis, providing that the proper contribution from GLN and ASN side chains to the 1667 and 1650 cm(-1) component bands has been taken into account. The standard deviation of the regression analysis for the per cent helix, extended, turn and irregular conformations was found to be 3.49%, 2.07%, 3.59% and 3.20%, respectively.