Catalytic Mechanism of Fatty Acid Photodecarboxylase: On the Detection and Stability of the Initial Carbonyloxy Radical Intermediate.

In fatty acid photodecarboxylase (FAP), light-induced formation of the primary radical product RCOO⋅ from fatty acid RCOO- occurs in 300 ps, upon which CO2 is released quasi-immediately. Based on the hypothesis that aliphatic RCOO⋅ (spectroscopically uncharacterized because unstable) absorbs in the red similarly to aromatic carbonyloxy radicals such as 2,6-dichlorobenzoyloxy radical (DCB⋅), much longer-lived linear RCOO⋅ has been suggested recently. We performed quantum chemical reaction pathway and spectral calculations. These calculations are in line with the experimental DCB⋅ decarboxylation dynamics and spectral properties and show that in contrast to DCB⋅, aliphatic RCOO⋅ radicals a) decarboxylate with a very low energetic barrier and on the timescale of a few ps and b) exhibit little red absorption. A time-resolved infrared spectroscopy experiment confirms very rapid, ≪300 ps RCOO⋅ decarboxylation in FAP. We argue that this property is required for the observed high quantum yield of hydrocarbons formation by FAP.

Chirped pulse upconversion for femtosecond mid-infrared spectroscopy at 100 kHz.

We demonstrate that chirped pulse up-conversion (CPU), a method routinely used with systems based on 1-kHz Titanium:Sapphire lasers, can be extended to a repetition rate of 100 kHz with an Ytterbium diode-pumped femtosecond amplifier. Individual mid-infrared spectra can thus be measured directly in the near infrared using a fast CMOS linescan camera. After an appropriate Fourier processing, a spectral resolution of 1.1 cm-1 is reported, currently limited by our spectrometer. Additionally, we demonstrate the application of CPU to a pump-probe measurement of the vibrational relaxation in carboxy-hemoglobin, and we show that the combination of fast scanning and fast acquisition enables a straightforward removal of pump scattering interference.

Phase-modulated rapid-scanning fluorescence-detected two-dimensional electronic spectroscopy.

We present a rapid-scanning approach to fluorescence-detected two-dimensional electronic spectroscopy that combines acousto-optic phase-modulation with digital lock-in detection. This approach shifts the signal detection window to suppress 1/f laser noise and enables interferometric tracking of the time delays to allow for correction of spectral phase distortions and accurate phasing of the data. This use of digital lock-in detection enables acquisition of linear and nonlinear signals of interest in a single measurement. We demonstrate the method on a laser dye, measuring the linear fluorescence excitation spectrum as well as rephasing, non-rephasing, and absorptive fluorescence-detected two-dimensional electronic spectra.

Electronic measurement of femtosecond time delays for arbitrary-detuning asynchronous optical sampling.

Arbitrary-Detuning ASynchronous OPtical Sampling (ADASOPS) is a pump-probe technique which relies on the stability of femtosecond oscillators. It provides access to a multiscale time window ranging up to millisecond, combined with a sub-picosecond time resolution. In contrast with the first ADASOPS demonstration based on the interferometric detection of coincidences between optical pulses, we show here that the optical setup can now be reduced to a mere pair of photodetectors embedded in a specially-designed electronic system. In analogy with super-resolution methods used in optical microscopy for localizing single emitters beyond the diffraction limit, we demonstrate that purely electronic means allow the determination of time delays between each pump-probe pulse pair with a standard deviation as small as 200 fs. The new method is shown to be simpler, more versatile and more accurate than the coincidence-based approach.

Multiscale control and rapid scanning of time delays ranging from picosecond to millisecond.

Femtosecond amplifiers seeded by two independent femtosecond oscillators normally produce amplified pulse pairs with a timing jitter equal to the oscillator period, which is typically around 12 ns for Titanium:Sapphire lasers. Combining Arbitrary-Detuning Asynchronous Optical Sampling (AD-ASOPS) with an appropriate selection of amplified pulses, we demonstrate that the time-delay distribution can be narrowed down to a 25-ps time window, allowing to produce spectral interference fringes for each amplified pulse pair. Subsequent AD-ASOPS determination of the actual time delay with subpicosecond accuracy allows to tailor the delay distribution with an electronic control all the way to the repetition period of the amplifiers. We thus demonstrate rapid scanning of the time delays up to nearly 1 ms with a sub-picosecond accuracy, which makes this method an ideal tool for multiscale pump-probe spectroscopy.

Transient Two-Dimensional Infrared Spectroscopy in a Vibrational Ladder.

We report on transient 2D Fourier transform infrared spectroscopy (2DIR) after vibrational ladder climbing induced in the CO-moiety longitudinal stretch of carboxyhemoglobin. The population distribution, spreading up to seven vibrational levels, results in a nonequilibrium 2DIR spectrum evidencing a large number of peaks that can be easily attributed to individual transitions thanks to the anharmonicity of the vibrational potential. We discuss the physical origin of the observed peaks as well as the qualitative behavior of the subsequent dynamics governed by population relaxation in the vibrational ladder.

Arbitrary-detuning asynchronous optical sampling with amplified laser systems.

We demonstrate that Arbitrary-Detuning ASynchronous OPtical Sampling (AD-ASOPS) makes possible multiscale pump-probe spectroscopy with time delays spanning from picosecond to millisecond. The implementation on pre-existing femtosecond amplifiers seeded by independent free-running oscillators is shown to be straightforward. The accuracy of the method is determined by comparison with spectral interferometry, providing a distribution with a standard deviation ranging from 0.31 to 1.7 ps depending on experimental conditions and on the method used to compute the AD-ASOPS delays.

Ultrafast Dynamics of Carboxy-Hemoglobin: Two-Dimensional Infrared Spectroscopy Experiments and Simulations.

This Letter presents a comparison between experimental and simulated 2D mid-infrared spectra of carboxy-hemoglobin in the spectral region of the carbon monoxide stretching mode. The simulations rely on a fluctuating potential energy surface that includes both the effect of heme and the protein surroundings computed from molecular dynamics simulations. A very good agreement between theory and experiment is obtained with no adjustable parameters. The simulations show that the effect of the distal histidine through the hydrogen bond is strong and is directly responsible for the slow decay of the frequency-frequency correlation function on a 10 ps time scale. This study confirms that fluctuations in carboxy-hemoglobin are more inhomogeneous than those in the more frequently studied carboxy-myoglobin. The comparison between simulations and experiments brings valuable information on the complex relation between protein structure and spectral diffusion.

Pulse shaping with birefringent crystals: a tool for quantum metrology.

A method for time differentiation based on a Babinet-Soleil-Bravais compensator is introduced. The complex transfer function of the device is measured using polarization spectral interferometry. Time differentiation of both the pulse field and pulse envelope are demonstrated over a spectral width of about 100 THz with a measured overlap with the objective mode greater than 99.8%. This pulse shaping technique is shown to be perfectly suited to time metrology at the quantum limit.

Arbitrary-detuning asynchronous optical sampling pump-probe spectroscopy of bacterial reaction centers.

A recently reported variant of asynchronous optical sampling compatible with arbitrary unstabilized laser repetition rates is applied to pump-probe spectroscopy. This makes possible the use of a 5.1 MHz chirped pulse oscillator as the pump laser, thus extending the available time window to almost 200 ns with a time resolution as good as about 320 fs. The method is illustrated with the measurement in a single experiment of the complete charge transfer dynamics of the reaction center from Rhodobacter sphaeroides.

Asynchronous optical sampling with arbitrary detuning between laser repetition rates.

A method of asynchronous optical sampling based on free-running lasers with no requirement on the repetition rates is presented. The method is based on the a posteriori determination of the delay between each pair of pulses. A resolution better than 400 fs over 13 ns total delay scan is demonstrated. In addition to the advantages of conventional asynchronous sampling techniques, this method allows a straightforward implementation on already-existing laser systems using a fiber-based setup and an appropriate acquisition procedure.

Strong ligand-protein interactions revealed by ultrafast infrared spectroscopy of CO in the heme pocket of the oxygen sensor FixL.

In heme-based sensor proteins, ligand binding to heme in a sensor domain induces conformational changes that eventually lead to changes in enzymatic activity of an associated catalytic domain. The bacterial oxygen sensor FixL is the best-studied example of these proteins and displays marked differences in dynamic behavior with respect to model globin proteins. We report a mid-IR study of the configuration and ultrafast dynamics of CO in the distal heme pocket site of the sensor PAS domain FixLH, employing a recently developed method that provides a unique combination of high spectral resolution and range and high sensitivity. Anisotropy measurements indicate that CO rotates toward the heme plane upon dissociation, as is the case in globins. Remarkably, CO bound to the heme iron is tilted by ~30° with respect to the heme normal, which contrasts to the situation in myoglobin and in present FixLH-CO X-ray crystal structure models. This implies protein-environment-induced strain on the ligand, which is possibly at the origin of a very rapid docking-site population in a single conformation. Our observations likely explain the unusually low affinity of FixL for CO that is at the origin of the weak ligand discrimination between CO and O(2). Moreover, we observe orders of magnitude faster vibrational relaxation of dissociated CO in FixL than in globins, implying strong interactions of the ligand with the distal heme pocket environment. Finally, in the R220H FixLH mutant protein, where CO is H-bonded to a distal histidine, we demonstrate that the H-bond is maintained during photolysis. Comparison with extensively studied globin proteins unveils a surprisingly rich variety in both structural and dynamic properties of the interaction of a diatomic ligand with the ubiquitous b-type heme-proximal histidine system in different distal pockets.

Impact of pulse polarization on coherent vibrational ladder climbing signals.

We report a theoretical study that elaborates the influence of the polarization state of both the pump and the probe pulse in ultrafast coherent vibrational ladder climbing experiments in the mid-infrared. Whereas a subensemble in a randomly oriented sample of molecules is excited by the pump pulse in this multiphoton process, further inhomogeneities such as the spatial profile of the laser beams, the longitudinal attenuation in the sample, and the probe beam polarization have to be taken into account. Analytical expressions for a density function describing the number of molecules that are exposed to an effective pump intensity are introduced, and the variation of the population distribution and the actual transient absorption signal in dependence on the polarization-state combinations for pump and probe pulse are discussed in detail. In simulations on the model system carboxy-hemoglobin, it is demonstrated that the polarization states play important roles both for exciting a certain population distribution and for actually observing it. In particular, it will be discussed under which conditions experimental data indicates a population inversion.

Direct mid-infrared femtosecond pulse shaping with a calomel acousto-optic programmable dispersive filter.

Direct amplitude and phase shaping of mid-infrared femtosecond pulses is realized with a calomel-based acousto-optic programmable dispersive filter transparent between 0.4 and 20 µm. The shaped pulse electric field is fully characterized with high accuracy, using chirped-pulse upconversion and time-encoded arrangement spectral phase interferometry for direct electric field reconstruction techniques. Complex mid-infrared pulse shapes at a center wavelength of 4.9 µm are generated with a spectral resolution of 14 cm(-1), which exceeds by a factor of 5 the reported experimental resolutions of calomel-based filters.

Dispersion-based pulse shaping for multiplexed two-photon fluorescence microscopy.

We demonstrate selective two-photon excited fluorescence microscopy with shaped pulses produced with a simple yet efficient scheme based on dispersive optical components. The pulse train from a broadband oscillator is split into two subtrains that are sent through different amounts of glass. Beam recombination results in pulse-shape switching at a rate of 150MHz. Time-resolved photon counting detection then provides two simultaneous images resulting from selective two-photon excitation, as demonstrated in a live embryo. Although less versatile than programmable pulse-shaping devices, this novel arrangement significantly improves the performance of selective microscopy using broadband shaped pulses while simplifying the experimental setup.

Suppression of perturbed free-induction decay and noise in experimental ultrafast pump-probe data.

We apply a Fourier filtering technique for the global removal of coherent contributions, like perturbed free-induction decay, and noise, to experimental pump-probe spectra. A further filtering scheme gains access to spectra otherwise only recordable by scanning the probe's center frequency with adjustable spectral resolution. These methods cleanse pump-probe data and allow improved visualization and simpler analysis of the contained dynamics. We demonstrate these filters using visible pump/mid-infrared probe spectroscopy of ligand dissociation in carboxyhemoglobin.

Unobtrusive interferometer tracking by path length oscillation for multidimensional spectroscopy.

We track the path difference between interferometer arms with few-nanometer accuracy without adding optics to the beam path. We measure the interference of a helium-neon beam that copropagates through the interferometer with midinfrared pulses used for multidimensional spectroscopy. This can indicate motion, but not direction. By oscillating the path length of one arm with a mirror on a piezoelectric stack and monitoring the oscillations of the recombined helium-neon beam, the direction can be calculated, and the path delay can be continuously tracked.

Multiplexed two-photon microscopy of dynamic biological samples with shaped broadband pulses.

Coherent control can be used to selectively enhance or cancel concurrent multiphoton processes, and has been suggested as a means to achieve nonlinear microscopy of multiple signals. Here we report multiplexed two-photon imaging in vivo with fast pixel rates and micrometer resolution. We control broadband laser pulses with a shaping scheme combining diffraction on an optically-addressed spatial light modulator and a scanning mirror allowing to switch between programmable shapes at kiloHertz rates. Using coherent control of the two-photon excited fluorescence, it was possible to perform selective microscopy of GFP and endogenous fluorescence in developing Drosophila embryos. This study establishes that broadband pulse shaping is a viable means for achieving multiplexed nonlinear imaging of biological tissues.

Femtosecond spectroscopy from the perspective of a global multidimensional response function.

At the microscopic level, multidimensional response functions, such as the nonlinear optical susceptibility or the time-ordered response function, are commonly used tools in nonlinear optical spectroscopy for determining the nonlinear polarization resulting from an arbitrary excitation. In this Account, we point out that the approach successfully developed for the nonlinear polarization can also be used in the case of a directly observable macroscopic quantity. This observable can be, for example, the electric field radiated in a nonlinear mixing experiment, the rate of fluorescence resulting from one- or two-photon absorption, or the rate of a photochemical reaction. For each of these physical processes, perturbation theory can be used to expand the measured quantity in a power series of the exciting field, and an appropriate global response function can be introduced for each order of perturbation. At order n, the multidimensional response function will depend on n variables (either time or frequency) and have the same general properties as the nonlinear susceptibility resulting, for example, from time invariance or causality. The global response function is introduced in this Account in close analogy with the nonlinear susceptibility or the time-ordered microscopic response. We discuss various applications of the global response function formalism. For example, it can be shown that in the weak field limit, a stationary signal induced in a time-invariant system is independent of the spectral phase of the exciting field. Although this result had been demonstrated previously, the global response function enables its derivation in a more general way because no specific microscopic model is needed. Multidimensional spectroscopy is obviously ideally suited to measure the global multidimensional response function. It is shown that the second (or third)-order response can be exactly measured with 2D (or 3D) spectroscopy by taking into account the exact shape of the exciting pulses. In the case of a 2D measurement of the third-order response, a particular projection of the complete 3D response function is actually measured. This projection can be related to a mixed time and frequency representation of the response function when the pulses are assumed to be infinitely short. We thus show that the global response function is a useful tool for deriving general results and that it should help in designing future experimental schemes for femtosecond spectroscopy.

Removing cross-phase modulation from midinfrared chirped-pulse upconversion spectra.

We observe that narrow spectral features in mid-infrared spectra obtained by chirped-pulse up-conversion are strongly distorted by cross-phase modulation between the mid-infrared field and the chirped pulse. We discuss the consequences of this effect on spectral resolution, and introduce a correction method that recovers masked lines. This simple correction can be applied either when the upconverted field is fully characterized, such as in multidimensional spectroscopy, or when causality can be used, such as in absorption spectroscopy, which we demonstrate experimentally.