Excited State Vibrational Dynamics Reveals a Photocycle That Enhances the Photostability of the TagRFP-T Fluorescent Protein.

High photostability is a desirable property of fluorescent proteins (FPs) for imaging, yet its molecular basis is poorly understood. We performed ultrafast spectroscopy on TagRFP and its 9-fold more photostable variant TagRFP-T (TagRFP S158T) to characterize their initial photoreactions. We find significant differences in their electronic and vibrational dynamics, including faster excited-state proton transfer and transient changes in the frequency of the v520 mode in the excited electronic state of TagRFP-T. The frequency of v520, which is sensitive to chromophore planarity, downshifts within 0.58 ps and recovers within 0.87 ps. This vibrational mode modulates the distance from the chromophore phenoxy to the side chain of residue N143, which we suggest can trigger cis/trans photoisomerization. In TagRFP, the dynamics of v520 is missing, and this FP therefore lacks an important channel for chromophore isomerization. These dynamics are likely to be a key mechanism differentiating the photostability of the two FPs.

Observation of Coherent Perfect Absorption in Oil Film on Water Surface and Sensitive Detection of Refractive Index Anisotropy in the Film.

Sharp reflection dips of 50% were observed when white light was incident from the side of a cell on a 1 µm thick film of silicone oil (polydimethylsiloxane, PDMS, nearly transparent in visible light, with the extinction coefficient κ ≈ 0.0001) above a water surface in the cell so that the total reflection condition was satisfied at the oil-air interface. This is the first observation of a coherent perfect absorption (CPA) phenomenon in liquid. The experimental results can be reproduced by the Fresnel reflectance of the monolayer film, but the wavelength positions at which the dip appears for s-polarized and p-polarized light are reversed if the refractive index of the oil film is assumed to be isotropic. The experimental results were correctly reproduced by assuming that the extraordinary-ray refractive index (light polarized perpendicular to the interface) is 1% larger than the ordinary-ray refractive index (light polarized parallel to the interface). This indicates that the polarization dependence of the CPA phenomenon is extremely sensitive to the difference between the in-plane and out-of-plane refractive indices of the thin film.

Revealing ultrafast vibronic dynamics of tetracene molecules with sub-8 fs UV impulsive Raman spectroscopy.

Ultrafast dynamics of tetracene molecules in THF solution were investigated using sub-8 fs ultraviolet pulse lasers and ab initio calculations. The time trace of absorbance changes exhibited ultrafast decay with a time constant of 165 ± 10 fs because of the relaxation from a vibronically hot excited state to the potential minimum in the S1 state. From the signals of absorbance changes in the negative time region, we obtained the electronic dephasing time of 31.27 ± 1.63 fs. Inverse Fourier transform of stationary absorption spectra exhibited rapid decay with 2.1 ± 0.08 fs. From these data, we estimated the ratio of total dephasing time to homogenous and inhomogeneous broadening as 6.7% and 93.3%, respectively. Impulsive Raman spectra reflect the wave packet dynamics of vibrational modes. Although inhomogeneous broadening blurred the phase jump across the resonance peak in the spectral range, 1156 and 1680 cm-1 vibrational modes exhibited a phase jump from -π to ∼π and -0.5π to ∼0.5π, respectively. The amplitude profiles of these vibrational modes agree with simulated vibronic progressions of combination bands. Time-frequency analysis revealed coupling dynamics between low- and high-frequency modes, where high-frequency modes are in-plane motions and low-frequency modes are out-of-plane motions. Therefore, these coupling dynamics induce symmetry-breaking of the molecular framework, which fastens the singlet fission process.

Extremely large electro-optic effect of TPPS J-aggregates in the PVA or PVP polymer matrix and aqueous solution.

The molecules of tetra-phenyl porphyrin tetra-sulfonic acid (TPPS) form a J-aggregate via self-organization in aqueous solution. The J-aggregates formed in an aqueous solution with added hydrochloric acid were dispersed in PVP polymer and subjected to electric field modulation spectroscopy. The observed difference in the static polarizability Δαp between the excited and ground states in the TPPS J-aggregates reached a value 2.66 × 104 times and 1.22 × 105 times larger than in the monomer molecules in PVA and PVP polymer matrices, respectively. This large enhancement factor of the electro-optic effect (EOE) may be induced by the higher-order structure of the J-aggregate. The different enhancement factor for each polymer matrix may be introduced by the structural differences between the polymer matrices. Electric field modulation spectroscopy of the J-aggregate in aqueous solution showed an absorbance change of 25% of the magnitude of absorbance peak.

Algorithm of auto-balancing noise-canceling based on noise correlation for high-speed balancing, high-dynamic range, and robustness against DC-offset drift.

The influence of the light source noise can be reduced by subtracting the signal of the light source noise (reference signal) from that of the probe light (probe signal). Here, it is essential that the intensities of the signals are equated. To equate the intensities, an auto-balancing method is widely employed, where the gain of the probe signal is feedback-controlled, regarding the DC component in the subtraction as an error signal. However, DC-offset drift causes a deviation from the optimal intensity balance. Additionally, the DC component is often several orders of magnitude larger than the sample signal, which requires a high-dynamic range in the circuitry. Furthermore, if the feedback control is too fast, it cancels out the sample signal. In this study, we formulate a noise correlation auto-balancing method, where the correlation of the reference signal and residual noise in the subtraction is employed as the error signal. With this scheme, all the above problems are avoided. The feasibility of the algorithm was demonstrated by a prototype circuitry and signals emulating the probe and reference signals. It did not suffer from the DC-offset drift, while a 44-dB canceling rate with auto-balancing of a 1.3-MHz cutoff frequency was demonstrated. We foresee, such as in pump/probe measurements, that this scheme improves the robustness, dynamic range, and response time required to follow changes in transmittance and the measurement position of the sample while employing a light source that is advantageous in wavelength selectivity, coherence, and cost but is noisy.

Advanced time-resolved absorption spectroscopy with an ultrashort visible/near IR laser and a multi-channel lock-in detector.

Ultrashort visible-near infrared (NIR) pulse generation and its applications to ultrafast spectroscopy are discussed. Femtosecond pulses of around 800 nm from a Ti:sapphire laser are used as a pump of an optical parametric amplifier (OPA) in a non-collinear configuration to generate ultrashort visible (500-780 nm) pulses and deep-ultraviolet (DUV, 259-282 nm) pulses. The visible-NIR pulses and DUV pulses were compressed to 3.9 fs and 10.4 fs, respectively, and used to elucidate various ultrafast dynamics in condensed matter with a sub-10 fs resolution by pump-probe measurements. We have also developed a 128-channel lock-in amplifier. The combined system of the world-shortest visible pulse from the OPA and the lock-in amplifier with the world-largest channel-number can clarify the sub-10 fs-dynamics in condensed matter. This system clarified structural changes in an excited state, reaction intermediate, and a transition state. This is possible even during molecular vibration and reactions via a real-time-resolved vibronic spectrum, which provides molecular structural change information. Also, ultrafast dynamics in exotic materials like carbon nanotubes, topological insulators, and novel solar battery systems have been clarified. Furthermore, the carrier-envelope phase in the ultrashort pulse has been controlled and measured.

Acute Spinal Cord Infarction with Preferential Involvement of Ventral Gray Matter: An Autopsy Report.

Herein, we report abdominal aortic thrombosis as a rare cause of acute spinal cord infarction. A 78-year-old man with multiple vascular risk factors developed acute paraplegia with sensory and urinary disturbances and signs of ischemia in both lower limbs. The post-mortem study done 3 days after the onset of symptoms revealed a large coagulum in the abdominal aorta, distal to the renal arteries and extending to bilateral common iliac arteries; in addition, marked atherosclerosis was present in most large blood vessels. Premature incomplete necrotic foci were seen in the ventral gray matter of the spinal cord from T6 through S5; the surrounding white matter and dorsal gray matter were spared. Considering our autopsy case, spinal cord gray matter may be more vulnerable to ischemia than the white matter.

Primary Electronic and Vibrational Dynamics of Cytochrome c Observed by Sub-10 fs NUV Laser Pulses.

The primary reaction mechanism of cytochrome c (Cyt c) was elucidated for two redox forms of ferric (oxidized) and ferrous (reduced) Cyt c by measuring their transient absorption (TA) spectra using a homemade sub-10 fs broadband NUV laser pulses system. The TA traces measured in the broad probe wavelength region were analyzed by the global analysis method to study the electronic dynamics. The difference of relaxation dynamics dependent on the excitation bandwidth enabled us to elucidate that the 2.5 ps component in ferrous Cyt c can be assigned to intramolecular vibration energy redistribution and not to vibrational cooling, which was not clear until this work. The temporal resolution of 10 fs observes TA signal modulation caused by the molecular vibration in the time domain, which can be used to calculate the instantaneous frequency of the molecular vibration mode. The observed vibrational dynamics has visualized that the heme structure changes in 0.8 ps for ferric Cyt c and in >1.0 ps for ferrous Cyt c. These estimated lifetimes of vibrational dynamics reflect vibrational relaxation in the ground state of ferric Cyt c and electronic transition from the S2 state to the S1 state in ferrous Cyt c, respectively.

Femtosecond time-evolution of mid-infrared spectral line shapes of Dirac fermions in topological insulators.

Mid-infrared (MIR) light sources have much potential in the study of Dirac-fermions (DFs) in graphene and topological insulators (TIs) because they have a low photon energy. However, the topological surface state transitions (SSTs) in Dirac cones are veiled by the free carrier absorption (FCA) with same spectral line shape that is always seen in static MIR spectra. Therefore, it is difficult to distinguish the SST from the FCA, especially in TIs. Here, we disclose the abnormal MIR spectrum feature of transient reflectivity changes (ΔR/R) for the non-equilibrium states in TIs, and further distinguish FCA and spin-momentum locked SST using time-resolved and linearly polarized ultra-broadband MIR spectroscopy with no environmental perturbation. Although both effects produce similar features in the reflection spectra, they produce completely different variations in the ΔR/R to show their intrinsic ultrafast dynamics.

Femtosecond electronic relaxation and real-time vibrational dynamics in 2'-hydroxychalcone.

Femtosecond ultrafast electronic relaxation and vibrational dynamics in 2'-hydroxychalcone after deep ultraviolet (DUV) excitation were observed by two types of pump-probe spectroscopy experiments, i.e., DUV-pump pulse and visible-broadband-probe pulse (DUV/Vis) experiments and DUV-pump and DUV-probe (DUV/DUV) pulse experiments. Time-dependent density functional theory (TDDFT) calculations were performed to elucidate relaxation dynamics from the third singlet electronic excited state S3. The DUV/Vis experiments and TDDFT calculations have disclosed the ultrafast dynamics of internal conversion from the initial S3 state (τ1 ≈ 35 fs) to the S1 state via a rapid process through the S3/S2 conical intersection and proton transfer [OH: τ2(H) ≈ 93 and OD: τ2(D) ≈ 164 fs] before deactivation through the S1/S0 conical intersection (τ3 ≈ 690 fs). Thanks to the ultrashort pump and probe pulses, real-time observation of vibrational modes coupled to the electronic excitation was realized providing both amplitudes and phases. Spectrogram analyses were performed based on the real-time spectra obtained by the DUV/Vis experiments, in which instantaneous vibrational frequencies reflecting molecular structural change after the impulsive excitation were visualized. The vibrational frequency of central C[double bond, length as m-dash]C bond stretch decreases from ∼1600 cm-1 to ∼1560 cm-1 in about 200-500 fs and recovers in ∼550 fs. Normal mode analyses along the decay path support the observed variation of the C[double bond, length as m-dash]C stretching frequency. The temporal weakening of the central C[double bond, length as m-dash]C bond is connected with the angle of the two aromatic rings which flip back to the initial conformation.

Exploring the ultrafast dynamics of a diarylethene derivative using sub-10 fs laser pulses.

A diarylethene derivative, 1,2-bis(2,4-dimethyl-5-phenyl-3-thienyl)perfluorocyclopentene (DMP), is a photoswitch molecule utilizing a reversible aromatic ring-opening reaction. The quantum yield of the ring-opening reaction is however remarkably low. We investigate the origin of this behaviour by means of ultrafast transient absorption spectroscopy utilizing sub-10 fs pulses, which is an invaluable tool for simultaneously studying both the electronic and the vibrational molecular dynamics. Namely, a noncollinear optical parametric amplifier (NOPA) generating sub-10 fs pulses in the spectral range 605-750 nm is employed. The transient absorption signal is modulated by several vibrational modes, which are compared with experimental and computational Raman spectra and then assigned to the ground or excited electronic state. We observe that the most pronounced vibrational mode - the ethylenic stretching mode at a frequency of 1501 cm-1 - exhibits instantaneous frequency and amplitude modulation. The observed modulations occur due to weak coupling with another 1431 cm-1 stretching mode mediated by a vibrational mode of low frequency, i.e. around 60 cm-1. Fast internal conversion S1 → S0 originates in a relaxation through a conical intersection (found by density-functional theory computations), facilitated by the two aforementioned stretching modes.

Label-Free Imaging of Melanoma with Confocal Photothermal Microscopy: Differentiation between Malignant and Benign Tissue.

Label-free confocal photothermal (CPT) microscopy was utilized for the first time to investigate malignancy in mouse skin cells. Laser diodes (LDs) with 405 nm or 488 nm wavelengths were used as pumps, and a 638 nm LD was used as a probe for the CPT microscope. A Grey Level Cooccurrence Matrix (GLCM) for texture analysis was applied to the CPT images. Nine GLCM parameters were calculated with definite definitions for the intracellular super-resolved CPT images, and the parameters Entropy, Contrast, and Variance were found to be most suited among the nine parameters to discriminate clearly between healthy cells and malignant cells when a 405 nm pump was used. Prominence, Variance, and Shade were most suited when a pump wavelength of 488 nm was used.

Utility of Autonomic Function Tests to Differentiate Dementia with Lewy Bodies and Parkinson Disease with Dementia from Alzheimer Disease.

OBJECTIVE: We studied autonomic disturbance in patients with dementia with Lewy bodies (DLB), Parkinson disease with dementia (PDD), Alzheimer disease (AD), to determine whether autonomic function tests can be used to distinguish these disorders. METHODS: Autonomic function was tested in 56 patients with DLB, 37 patients with PDD, and 59 patients with AD by using the sympathetic skin response, coefficient of variation in R-R interval, the head-up tilt test, serum norepinephrine concentration, and 123I-meta-iodobenzylguanidine cardiac scintigraphy. Symptoms of autonomic dysfunction, such as constipation, urinary symptoms, and orthostatic hypotension, were also noted. RESULTS: The groups did not differ on baseline characteristics other than those associated with Parkinsonism and dementia. All patients with DLB and PDD had some dysautonomia, whereas rates were much lower for patients with AD (19%). Significantly more DLB and PDD patients than AD patients showed abnormalities on autonomic function tests. CONCLUSIONS: Autonomic function tests might be quite useful to distinguish DLB and PDD from AD.

Ultrafast dynamics of multi-exciton state coupled to coherent vibration in zinc chlorin aggregates for artificial photosynthesis.

Ultrafast vibronic dynamics induced by the interaction of the Frenkel exciton with the coherent molecular vibrations in a layer-structured zinc chlorin aggregates prepared for artificial photosynthesis have been studied by 7.1 fs real-time vibrational spectroscopy with multi-spectrum detection. The fast decay of 100 ± 5fs is ascribed to the relaxation from the higher multi-exciton state (MES) to the one-exciton state, and the slow one of 863 ± 70fs is assigned to the relaxation from Q-exciton state to the dark nonfluorescent charge-transfer (CT) state, respectively. In addition, the wavelength dependences of the exciton-vibration coupling strength are found to follow the zeroth derivative of the transient absorption spectra of the exciton. It could be explained in term of the transition dipole moment modulated by dynamic intensity borrowing between the B transition and the Q transition through the vibronic interactions.

Photothermal imaging of skeletal muscle mitochondria.

The morphology and topology of mitochondria provide useful information about the physiological function of skeletal muscle. Previous studies of skeletal muscle mitochondria are based on observation with transmission, scanning electron microscopy or fluorescence microscopy. In contrast, photothermal (PT) microscopy has advantages over the above commonly used microscopic techniques because of no requirement for complex sample preparation by fixation or fluorescent-dye staining. Here, we employed the PT technique using a simple diode laser to visualize skeletal muscle mitochondria in unstained and stained tissues. The fine mitochondrial network structures in muscle fibers could be imaged with the PT imaging system, even in unstained tissues. PT imaging of tissues stained with toluidine blue revealed the structures of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria and the swelling behavior of mitochondria in damaged muscle fibers with sufficient image quality. PT image analyses based on fast Fourier transform (FFT) and Grey-level co-occurrence matrix (GLCM) were performed to derive the characteristic size of mitochondria and to discriminate the image patterns of normal and damaged fibers.

Schiff Base Proton Acceptor Assists Photoisomerization of Retinal Chromophores in Bacteriorhodopsin.

In this study, we investigated the ultrafast dynamics of bacteriorhodopsins (BRs) from Haloquadratum walsbyi (HwBR) and Haloarcula marismortui (HmBRI and HmBRII). First, the ultrafast dynamics were studied for three HwBR samples: wild-type, D93N mutation, and D104N mutation. The residues of the D93 and D104 mutants correspond to the control by the Schiff base proton acceptor and donor of the proton translocation subchannels. Measurements indicated that the negative charge from the Schiff base proton acceptor residue D93 interacts with the ultrafast and substantial change of the electrostatic potential associated with chromophore isomerization. By contrast, the Schiff base proton donor assists the restructuring of the chromophore cavity hydrogen-bond network during the thermalization of the vibrational hot state. Second, the ultrafast dynamics of the wild-types of HwBR, HmBRI, and HmBRII were compared. Measurements demonstrated that the hydrogen-bond network in the extracellular region in HwBR and HmBRII slows the photoisomerization of retinal chromophores, and the negatively charged helices on the cytoplasmic side of HwBR and HmBRII accelerate the thermalization of the vibrational hot state of retinal chromophores. The similarity of the correlation spectra of the wild-type HmBRI and D104N mutant of HwBR indicates that inactivation of the Schiff base proton donor induces a positive charge on the helices of the cytoplasmic side.

Gray-level co-occurrence matrix analysis of several cell types in mouse brain using resolution-enhanced photothermal microscopy.

Qualifications of intracellular structure were performed for the first time using the gray-level co-occurrence matrix (GLCM) method for images of cells obtained by resolution-enhanced photothermal imaging. The GLCM method has been used to extract five parameters of texture features for five different types of cells in mouse brain; pyramidal neurons and glial cells in the basal nucleus (BGl), dentate gyrus granule cells, cerebellar Purkinje cells, and cerebellar granule cells. The parameters are correlation, contrast, angular second moment (ASM), inverse difference moment (IDM), and entropy for the images of cells of interest in a mouse brain. The parameters vary depending on the pixel distance taken in the analysis method. Based on the obtained results, we identified that the most suitable GLCM parameter is IDM for pyramidal neurons and BGI, granule cells in the dentate gyrus, Purkinje cells and granule cells in the cerebellum. It was also found that the ASM is the most appropriate for neurons in the basal nucleus.

Ultrafast carrier dynamics in Ge by ultra-broadband mid-infrared probe spectroscopy.

In this study, we carried out 800-nm pump and ultra-broadband mid-infrared (MIR) probe spectroscopy with high time-resolution (70 fs) in bulk Ge. By fitting the time-resolved difference reflection spectra [ΔR(ω)/R(ω)] with the Drude model in the 200-5000 cm-1 region, the time-dependent plasma frequency and scattering rate have been obtained. Through the calculation, we can further get the time-dependent photoexcited carrier concentration and carrier mobility. The Auger recombination essentially dominates the fast relaxation of photoexcited carriers within 100 ps followed by slow relaxation due to diffusion. Additionally, a novel oscillation feature is clearly found in time-resolved difference reflection spectra around 2000 cm-1 especially for high pump fluence, which is the Lorentz oscillation lasting for about 20 ps due to the Coulomb force exerted just after the excitation.

Surface-Enhanced Impulsive Coherent Vibrational Spectroscopy.

Surface-enhanced Raman spectroscopy (SERS) has attracted a lot of attention in molecular sensing because of the remarkable ability of plasmonic metal nanostructures to enhance the weak Raman scattering process. On the other hand, coherent vibrational spectroscopy triggered by impulsive excitation using ultrafast laser pulses provides complete information about the temporal evolution of molecular vibrations, allowing dynamical processes in molecular systems to be followed in "real time". Here, we combine these two concepts and demonstrate surface-enhanced impulsive vibrational spectroscopy. The vibrational modes of the ground and excited states of poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV), spin-coated on a substrate covered with monodisperse silver nanoparticles, are impulsively excited with a sub-10 fs pump pulse and characterized with a delayed broad-band probe pulse. The maximum enhancement in the spectrally and temporally resolved vibrational signatures averaged over the whole sample is about 4.6, while the real-time information about the instantaneous vibrational amplitude together with the initial vibrational phase is preserved. The phase is essential to determine the vibrational contributions from the ground and excited states.