Two-color Experiments Research Articles

Serial femtosecond crystallography opens new avenues for Structural Biology.

Free electron lasers (FELs) provide X-ray pulses in the femtosecond time domain with up to 10(12) higher photon flux than synchrotrons and open new avenues for the determination of difficult to crystallize proteins, like large complexes and human membrane proteins. While the X-ray pulses are so strong that they destroy any solid material, the crystals diffract before they are destroyed. The most successful application of FELs for biology has been the method of serial femtosecond crystallography (SFX) where nano or microcrystals are delivered to the FEL beam in a stream of their mother liquid at room temperature, which ensures the replenishment of the sample before the next X-ray pulse arrives. New injector technology allows also for the delivery of crystal in lipidic cubic phases or agarose, which reduces the sample amounts for an SFX data set by two orders of magnitude. Time-resolved SFX also allows for analysis of the dynamics of biomolecules, the proof of principle being recently shown for light-induced reactions in photosystem II and photoactive yellow protein. An SFX data sets consist of thousands of single crystal snapshots in random orientations, which can be analyzed now "on the fly" by data analysis programs specifically developed for SFX, but de-novo phasing is still a challenge, that might be overcome by two-color experiments or phasing by shape transforms.

Precise Control and Measurement of Temperature with Femtosecond Optical Tweezers

Optical traps have often been used for physical manipulation and transport within liquids for studying bio-systems. In this connection, though a lot of work has focused on the property of the trapped particle, there is little effort on utilizing the effect of the trapping environment. Here we demonstrate a novel method for exploiting the effect of trapping environment in observing the temperature rise in liquids directly at the vicinity of an optical trap center. Our approach utilizes the photo-thermal effect at micro-volume dimension to measure temperature, which could eventually be extended to in-vivo conditions. Our two-color experiment is a unique combination of a non-heating femtosecond trapping laser at 780 nm, which is coupled to a femtosecond infrared heating laser at 1560 nm. Femtosecond pulsed laser heating is much more effective than the traditional continuous-wave laser, which increases the sensitivity of our measurements. The heating laser precisely controls temperature at the focal volume of the trap center using low powers at high repetition rate. The changed values of corner frequency of the optical trap due to this local heating is recognized from its power spectra. The solvent having hydroxyl group is very sensitive to 1560 nm laser due to non-radiative relaxation from their higher excited state. Since most bio-systems contain hydroxyl group, they would be highly responsive to our heating laser, while our trapping laser is highly transparent for such systems. Our method could be used to specifically heat a particular cell to evaluate its faster cell division and also can be utilized for the possible phototherapy of cancer cells. The advantage of this technique over the conventional phototherapy is that this is highly localized due to focusing through high numerical aperture resulting in a high gradient of temperature.

Helium Tagging Infrared Photodissociation Spectroscopy of Reactive Ions.

The interrogation of reaction intermediates is key for understanding chemical reactions; however their direct observation and study remains a considerable challenge. Mass spectrometry is one of the most sensitive analytical techniques, and its use to study reaction mixtures is now an established practice. However, the information that can be obtained is limited to elemental analysis and possibly to fragmentation behavior, which is often challenging to analyze. In order to extend the available experimental information, different types of spectroscopy in the infrared and visible region have been combined with mass spectrometry. Spectroscopy of mass selected ions usually utilizes the powerful sensitivity of mass spectrometers, and the absorption of photons is not detected as such but rather translated to mass changes. One approach to accomplish such spectroscopy involves loosely binding a tag to an ion that will be removed by absorption of one photon. We have constructed an ion trapping instrument capable of reaching temperatures that are sufficiently low to enable tagging by helium atoms in situ, thus permitting infrared photodissociation spectroscopy (IRPD) to be carried out. While tagging by larger rare gas atoms, such as neon or argon is also possible, these may cause significant structural changes to small and reactive species, making the use of helium highly beneficial. We discuss the "innocence" of helium as a tag in ion spectroscopy using several case studies. It is shown that helium tagging is effectively innocent when used with benzene dications, not interfering with their structure or IRPD spectrum. We have also provided a case study where we can see that despite its minimal size there are systems where He has a huge effect. A strong influence of the He tagging was shown in the IRPD spectra of HCCl(2+) where large spectral shifts were observed. While the presented systems are rather small, they involve the formation of mixtures of isomers. We have therefore implemented two-color experiments where one laser is employed to selectively deplete a mixture by one (or more) isomer allowing helium tagging IRPD spectra of the remaining isomer(s) to be recorded via the second laser. Our experimental setup, based on a linear wire quadrupole ion trap, allows us to deplete almost 100% of all helium tagged ions in the trap. Using this special feature, we have developed attenuation experiments for determination of absolute photofragmentation cross sections. At the same time, this approach can be used to estimate the representation of isomers in a mixture. The ultimate aim is the routine use of this instrument and technique to study a wide range of reaction intermediates in catalysis. To this end, we present a study of hypervalent iron(IV)-oxo complexes ([(L)Fe(O)(NO3)](+)). We show that we can spectroscopically differentiate iron complexes with S = 1 and S = 2 according to the stretching vibrations of a nitrate counterion.

Modeling blue to UV upconversion in β-NaYF4:Tm3.

Samples of 0.01% and 0.3% Tm3+-doped β-NaYF4 show upconverted UV luminescence at 27 660 cm-1 (361 nm) after blue excitation at 21 140 cm-1 (473 nm). Contradictory upconversion mechanisms in the literature are reviewed and two of them are investigated in detail. Their agreement with emission and two-color excitation experiments is examined and compared. Decay curves are analyzed using the Inokuti-Hirayama model, an average rate equation model, and a microscopic rate equation model that includes the correct extent of energy transfer. Energy migration is found to be negligible in these samples, and hence the average rate equation model fails to correctly describe the decay curves. The microscopic rate equation model accurately fits the experimental data and reveals the strength and multipolarity of various interactions. This microscopic model is able to determine the most likely upconversion mechanism.

A Multicolor Single-Molecule FRET Approach to Study Protein Dynamics and Interactions Simultaneously.

Single-molecule Förster resonance energy transfer (smFRET) is a versatile tool for studying biomolecules in a quantitative manner. Multiple conformations within and interactions between biomolecules can be detected and their kinetics can be determined. Thus, smFRET has become an essential tool in enzymology. Ordinary two-color smFRET experiments can provide only limited insight into the function of biological systems, which commonly consist of more than two components. A complete understanding of complex multicomponent biological systems requires correlated information on conformational rearrangements on the one hand and transient interactions with binding partners on the other. Multicolor smFRET experiments enable the direct observation of such correlated dynamics and interactions. Here we demonstrate the power and limitations of multicolor smFRET experiments including the description of a multicolor smFRET setup and data analysis. A general analytical procedure for multicolor smFRET data is presented and applied to the multicomponent heat shock protein 90 system. This allows us to identify microscopic states in transient complexes. Conformational dynamics and nucleotide binding are simultaneously detected, which is impossible using two-color smFRET. Additionally, their correlation is quantified using 3D ensemble hidden Markov analysis, in and out of equilibrium. This method is perfectly suited for protein systems that are much more sophisticated than previously studied DNA-based systems. By extending the application to biologically relevant systems, multicolor smFRET comes of age and provides a unique mechanistic insight into protein machines.

Dynamics of the formation of laser-induced periodic surface structures (LIPSS) upon femtosecond two-color double-pulse irradiation of metals, semiconductors, and dielectrics

In order to address the dynamics and physical mechanisms of LIPSS formation for three different classes of materials (metals, semiconductors, and dielectrics), two-color double-fs-pulse experiments were performed on Titanium, Silicon and Fused Silica. For that purpose a Mach–Zehnder interferometer generated polarization controlled (parallel or cross-polarized) double-pulse sequences at 400nm and 800nm wavelength, with inter-pulse delays up to a few picoseconds. Multiple of these two-color double-pulse sequences were collinearly focused by a spherical mirror to the sample surfaces. The fluence of each individual pulse (400nm and 800nm) was always kept below its respective ablation threshold and only the joint action of both pulses lead to the formation of LIPSS. Their resulting characteristics (periods, areas) were analyzed by scanning electron microscopy. The periods along with the LIPSS orientation allow a clear identification of the pulse which dominates the energy coupling to the material. For strong absorbing materials (Silicon, Titanium), a wavelength-dependent plasmonic mechanism can explain the delay-dependence of the LIPSS. In contrast, for dielectrics (Fused Silica) the first pulse always dominates the energy deposition and LIPSS orientation, supporting a non-plasmonic formation scenario. For all materials, these two-color experiments confirm the importance of the ultrafast energy deposition stage for LIPSS formation.

Theoretical Study of Ultrafast Electron Dynamics in Amino Acids

SynopsisPrompt ionization of large biological molecules may induce ultrafast charge migration along the molecular skeleton, preceding any nuclear rearrangement. This phenomenon has been recently observed in the amino acid phenylalanine in a two-color pump probe experiment, where the production of ionic fragments was measured as a function of the time delay between the two pulses and charge fluctuations manifested as sub-4.5 fs oscillations in the quantum yield of a specific doubly charged fragment. We present our latest results in glycine, and compare with previous findings in phenylalanine. We seek to perform a systematic study including larger aminoacids such as tryptophan.

A Simplified Method for Identification of the Vibrational Series of Long-Range States in Na2

We performed two-color photoassociative ionization experiments in cold sodium atoms in a dark MOT. We produce a sample where most of the atoms were in the ground hyperfine state F=1. Applying a laser with variable frequency, in the region of 0 to −32 GHz, we obtained a photoassociative spectra containing sequences of different long-range potentials. Using the scaling law for intermediate states, we identified the vibrational series of involved states, separating the sequences. In this way, we have identified the vibrational series for the $1_{g}, 0_{u}^{+}$ , and $0_{g}^{-}$ states contributions.

A simple method for GFP- and RFP-based dual color single-molecule localization microscopy.

The recent development of single-molecule localization-based super-resolution techniques has afforded a resolution in the nanometer range in light microscopy. The ability to resolve biological structures on this scale by multicolor techniques faces significant challenges which have prevented their widespread use. Here, we provide a generic approach for high-quality simultaneous two-color single-molecule localization microscopy imaging of any combination of GFP- and RFP-tagged proteins with the use of nanobodies. Our method addresses a number of common issues related to two-color experiments, including accuracy and density of labeling as well as chromatic aberration and color-crosstalk with only minimal technical requirements. We demonstrate two-color imaging of various nanoscopic structures and show a compound resolution down to the limit routinely achieved only in a single color.

Femtosecond laser-induced periodic surface structures on silicon upon polarization controlled two-color double-pulse irradiation.

Two-color double-fs-pulse experiments were performed on silicon wafers to study the temporally distributed energy deposition in the formation of laser-induced periodic surface structures (LIPSS). A Mach-Zehnder interferometer generated parallel or cross-polarized double-pulse sequences at 400 and 800 nm wavelength, with inter-pulse delays up to a few picoseconds between the sub-ablation 50-fs-pulses. Multiple two-color double-pulse sequences were collinearly focused by a spherical mirror to the sample. The resulting LIPSS characteristics (periods, areas) were analyzed by scanning electron microscopy. A wavelength-dependent plasmonic mechanism is proposed to explain the delay-dependence of the LIPSS. These two-color experiments extend previous single-color studies and prove the importance of the ultrafast energy deposition for LIPSS formation.

Mapping energy transfer channels in fucoxanthin–chlorophyll protein complex

Fucoxanthin–chlorophyll protein (FCP) is the key molecular complex performing the light-harvesting function in diatoms, which, being a major group of algae, are responsible for up to one quarter of the total primary production on Earth. These photosynthetic organisms contain an unusually large amount of the carotenoid fucoxanthin, which absorbs the light in the blue–green spectral region and transfers the captured excitation energy to the FCP-bound chlorophylls. Due to the large number of fucoxanthins, the excitation energy transfer cascades in these complexes are particularly tangled. In this work we present the two-color two-dimensional electronic spectroscopy experiments on FCP. Analysis of the data using the modified decay associated spectra permits a detailed mapping of the excitation frequency dependent energy transfer flow with a femtosecond time resolution.

Wavefront-correction for nearly diffraction-limited focusing of dual-color laser beams to high intensities.

We demonstrate wavefront correction of terawatt-peak-power laser beams at two distinct and well-separated wavelengths. Simultaneous near diffraction-limited focusability is achieved for both the fundamental (800 nm) and second harmonic (400 nm) of Ti:sapphire-amplified laser light. By comparing the relative effectiveness of various correction loops, the optimal ones are found. Simultaneous correction of both beams of different color relies on the linear proportionality between their wavefront aberrations. This method can enable two-color experiments at relativistic intensities.

Laser-induced periodic surface structures on fused silica upon cross-polarized two-color double-fs-pulse irradiation

The dynamics of the formation of laser-induced periodic surface structures (LIPSS) on fused silica upon irradiation with linearly polarized fs-laser pulses (50 fs pulse duration) is studied by cross-polarized two-color double-fs-pulse experiments. In order to analyze the relevance of temporally distributed energy deposition in the early stage of LIPSS formation, a Mach-Zehnder interferometer was used for generating multiple double-pulse sequences at two different wavelengths (400 and 800nm). The inter-pulse delay between the individual cross-polarized pulses of each sequence was systematically varied in the sub-ps range and the resulting LIPSS morphologies were characterized by scanning electron microscopy. It is found that the polarization of the first laser pulse arriving to the surface determines the orientation and the periodicity of the LIPSS. These two-color experiments further confirm the importance of the ultrafast energy deposition to the silica surface for LIPSS formation, particularly by the first laser pulse of each sequence. The second laser pulse subsequently reinforces the previously seeded spatial LIPSS characteristics (period, orientation).

Crossed excitons in a semiconductor nanostructure of mixed dimensionality

Semiconductor systems of reduced dimensionality, e.g., quantum dots or quantum wells, display a characteristic spectrum of confined excitons. Combining several of these systems may lead to the formation of “crossed” excitons, and thus new equilibrium states and scattering channels. We derive gain excitation spectra from two-color pump-probe experiments on an In(Ga)As based quantum dot semiconductor optical amplifier by analyzing the amplitudes of the traces. This grants access to the quantum dot response, even in the presence of strong absorption by the surroundings at the excitation energy. The gain excitation spectra yield evidence of crossed quantum dot-bulk states.

Ultrafast vibrational dynamics of BH4(-) ions in liquid and crystalline environments.

Ultrafast vibrational dynamics of BH4(-) ions, the key units in boron hydride materials for hydrogen storage, are studied in diluted polar liquid solution and in NaBH4 crystallites by femtosecond infrared spectroscopy. Two-color pump-probe experiments reveal v = 1 lifetimes of 3 ps for the asymmetric BH4(-) stretching mode ν3 and of 3.6 ps for the asymmetric bending mode ν4 in the solvent isopropylamine. We provide direct evidence for the BH4(-) stretching relaxation pathway via the asymmetric bending mode ν4 by probing the latter after femtosecond excitation of ν3. Pump-probe traces measured in the crystalline phase show signatures of radiative coupling between the densely packed BH4(-) oscillators, most clearly manifested in an accelerated subpicosecond depopulation of the v = 1 state of the ν4 mode. The radiative decay is followed by incoherent vibrational relaxation similar to the liquid phase. The excess energy released in the relaxation processes of the BH4(-) intramolecular modes is transferred into the environment with thermal pump-probe signals being much more pronounced in the dense solid than in the diluted solution.

Half-cycle pulses in the mid-infrared from a two-color laser-induced filament

Four-wave mixing (FWM) of femtosecond near-infrared laser pulses and its second harmonic in the filamentation regime is shown to give rise to ultrashort field waveforms in the mid-infrared with pulse widths as short as a half of the field cycle and produce ultrabroadband supercontinuum spectra stretching from the mid-IR to the terahertz region. Generation of 7-fs pulses centered at 4.35 μm is demonstrated by a two-color filamentation experiment, where the 25-fs, 800-nm fundamental-wavelength output of a Ti: Sapphire laser is mixed with its second harmonic. The spectral and temporal properties of the mid-IR waveforms, as well as their emission pattern, are consistent with the FWM scenario of frequency conversion generalized to include the Kerr effect and ionization-induced refractive-index modulation.

Gain dynamics of quantum dot devices for dual-state operation

Ground state gain dynamics of In(Ga)As-quantum dot excited state lasers are investigated via single-color ultrafast pump-probe spectroscopy below and above lasing threshold. Two-color pump-probe experiments are used to localize lasing and non-lasing quantum dots within the inhomogeneously broadened ground state. Single-color results yield similar gain recovery rates of the ground state for lasing and non-lasing quantum dots decreasing from 6 ps to 2 ps with increasing injection current. We find that ground state gain dynamics are influenced solely by the injection current and unaffected by laser operation of the excited state. This independence is promising for dual-state operation schemes in quantum dot based optoelectronic devices.

Nonlinear spectroscopy in the near-field: time resolved spectroscopy and subwavelength resolution non-invasive imaging

AbstractThe combination of near-field microscopy along with nonlinear optical spectroscopic techniques is presented here. The scanning near-field imaging technique can be integrated with nonlinear spectroscopic techniques to improve spatial and axial resolution of the images. Additionally, ultrafast dynamics can be probed down to nano-scale dimension. The review shows some examples for this combination, which resulted in an exciton map and vibrational contrast images with sub-wavelength resolution. Results of two-color femtosecond time-resolved pump-probe experiments using scanning near-field optical microscopy (SNOM) on thin films of the organic semiconductor 3,4,9,10 Perylenetetracarboxylic dianhydride (PTCDA) are presented. While nonlinear Raman techniques have been used to obtain highly resolved images in combination with near-field microscopy, the use of femtosecond laser pulses in electronic resonance still constitutes a big challenge. Here, we present our first results on coherent anti-Stokes Raman scattering (fs-CARS) with femtosecond laser pulses detected in the near-field using SNOM. We demonstrate that highly spatially resolved images can be obtained from poly(3-hexylthiophene) (P3HT) nano-structures where the fs-CARS process was in resonance with the P3HT absorption and with characteristic P3HT vibrational modes without destruction of the samples. Sub-diffraction limited lateral resolution is achieved. Especially the height resolution clearly surpasses that obtained with standard microCARS. These results will be the basis for future investigations of mode-selective dynamics in the near-field.

Strong Terahertz Emission and Its Origin from Catalyst-Free InAs Nanowire Arrays

The unique features of nanowires (NW), such as the high aspect ratio and extensive surface area, are expected to play a key role in the development of very efficient semiconductor surface emitters in the terahertz (THz) spectral range. Here, we report on optically excited THz emission from catalyst-free grown arrays of intrinsically n-type InAs NWs using THz time-domain spectroscopy. Depending on the aspect ratio, the THz emission efficiency of the n-type InAs NWs is found to be up to ∼3 times stronger than that of bulk p-type InAs, known as currently the most efficient semiconductor-based THz surface emitter. Characteristic differences from bulk p-type InAs are particularly revealed from excitation wavelength-dependent measurements, showing monotonously increasing THz pulse amplitude in the NW arrays with increasing photon energy. Further polarization-dependent and two-color pump-probe experiments elucidate the physical mechanism of the THz emission: In contrast to bulk p-type InAs, where the anisotropic photoconductivity in the surface electric field is the dominant cause for THz pulse generation, the origin of the intrinsic THz emission in the NWs is based on the photo-Dember effect. The strong THz emission from high aspect ratio NW arrays further suggests an improved out-coupling of the radiation, while further enhancements in efficiency using core-shell NW geometries are discussed.

Glycan Catabolism by Human Gut Symbionts involves Dynamic Protein Interactions

The human gut hosts trillions of bacteria that directly influence human health. The majority of gut microbiota play an important role in nutrition by metabolizing host-indigestible complex glycans into short-chain fatty acids. Bacteroides thetaiotaomicron (Bt), a prominent bacterial symbiont in the distal gut, metabolizes over a dozen complex glycans using membrane-associated protein complexes. The Starch Utilization System (Sus), a multi-protein complex in Bt that is essential for growth on starch, uses eight proteins (SusRABCDEFG) to process starch. SusCDEFG localize in the outer membrane and likely form a complex to facilitate starch binding, degradation and import. However, conventional biochemical methods have been unable to completely reveal the assembly and dynamics of these proteins in response to starch. We have applied single-molecule super-resolution imaging to characterize the Sus complex response to different sugars in live Bt under anaerobic conditions. Protein correlation studies performed with HaloTag-labeled SusG and other fluorescent antibody-labeled Sus proteins demonstrated that simple sugars such as glucose or maltose do not induce Sus complex assembly. Conversely, incubation of Bt cells with starch enhanced the co-localization of Sus proteins, suggesting starch-induced assembly of the complex. Furthermore, single-molecule tracking revealed that in the absence of starch, SusG predominately moves in a fast diffusion mode corresponding to freely diffusing SusG. In contrast, two-color single-molecule experiments performed with fluorophore-labeled starch and SusG showed that starch confines the free motion of SusG and induces the assembly of Sus proteins. Overall, our results suggest that starch catabolism involves dynamic interactions of Sus proteins, which assemble as a complex in the presence of starch in live cells.