Ultrafast Transient Absorption Spectra and Kinetics of Rod and Cone Visual Pigments.

Rods and cones are the photoreceptor cells containing the visual pigment proteins that initiate visual phototransduction following the absorption of a photon. Photon absorption induces the photochemical transformation of a visual pigment, which results in the sequential formation of distinct photo-intermediate species on the femtosecond to millisecond timescales, whereupon a visual electrical signal is generated and transmitted to the brain. Time-resolved spectroscopic studies of the rod and cone photo-intermediaries enable the detailed understanding of initial events in vision, namely the key differences that underlie the functionally distinct scotopic (rod) and photopic (cone) visual systems. In this paper, we review our recent ultrafast (picoseconds to milliseconds) transient absorption studies of rod and cone visual pigments with a detailed comparison of the transient molecular spectra and kinetics of their respective photo-intermediaries. Key results include the characterization of the porphyropsin (carp fish rhodopsin) and human green-cone opsin photobleaching sequences, which show significant spectral and kinetic differences when compared against that of bovine rhodopsin. These results altogether reveal a rather strong interplay between the visual pigment structure and its corresponding photobleaching sequence, and relevant outstanding questions that will be further investigated through a forthcoming study of the human blue-cone visual pigment are discussed.

Enhancing the upconversion efficiency of NaYF4:Yb,Er microparticles for infrared vision applications.

In this study, (NaYF4:Yb,Er) microparticles dispersed in water and ethanol, were used to generate 540 nm visible light from 980 nm infrared light by means of a nonlinear stepwise two-photon process. IR-reflecting mirrors placed on four sides of the cuvette that contained the microparticles increased the intensity of the upconverted 540 nm light by a factor of three. We also designed and constructed microparticle-coated lenses that can be used as eyeglasses, making it possible to see rather intense infrared light images that are converted to visible.

Ultrafast spectra and kinetics of human green-cone visual pigment at room temperature.

Rhodopsin is the pigment that enables night vision, whereas cone opsins are the pigments responsible for color vision in bright-light conditions. Despite their importance for vision, cone opsins are poorly characterized at the molecular level compared to rhodopsin. Spectra and kinetics of the intermediate states of human green-cone visual pigment (mid-wavelength sensitive, or MWS opsin) were measured and compared with the intermediates and kinetics of bovine rhodopsin. All the major intermediates of the MWS opsin were recorded in the picosecond to millisecond time range. Several intermediates in MWS opsin appear to have characteristics similar to the intermediates of bovine rhodopsin; however, there are some marked differences. One of the most striking differences is in their kinetics, where the kinetics of the MWS opsin intermediates are slower compared to those of the bovine rhodopsin intermediates.

Comparison of Bovine and Carp Fish Visual Pigment Photo-Intermediates at Room Temperature.

This paper presents room temperature nanoseconds to milliseconds time-resolved spectra and kinetics of the intermediate states and species of bovine and carp fish rhodopsin visual pigments, which also contained ~5% cone pigments. The nanoseconds to milliseconds range cover all the major intermediates in the visual phototransduction process except the formation of bathorhodopsin intermediate which occurs at the femtosecond time scale. The dynamics of these visual pigment intermediates are initiated by excitation with a 532 nm nanosecond laser pulse. The recorded differences between bovine and carp rhodopsin time-resolved spectra of the formation and decay kinetics of their intermediates are presented and discussed. The data show that the carp samples batho intermediate decays faster, nearly by a factor of three, compared to the bovine samples. The formation and decay spectra and kinetics of rhodopsin outer segments and extracted rhodopsin inserted in buffer solution were found to be identical, with very small differences between them in the decay lifetimes of bathorhodopsin and formation of lumirhodopsin.

Cell-phone camera Raman spectrometer.

In this report, we describe the design, construction, and operation of a cell-phone-based Raman and emission spectral detector, which when coupled to a diffraction grating and cell-phone camera system provides means for the detection, recording, and identification of chemicals, drugs, and biological molecules, in situ by means of their Raman and fluorescence spectra. The newly constructed cell-phone spectrometer system was used to record Raman spectra from various chemicals and biological molecules including the resonance enhanced Raman spectra of carrots and bacteria. In addition, we present the quantitative analysis of alcohol-water Raman spectra, performed using our cell-phone spectrometer. The designed and constructed system was also used for constructing Raman images of the samples by utilizing a position scanning stage in conjunction with the system. This compact and portable system is well suited for in situ field applications of Raman and fluorescence spectroscopy and may also be an integrated feature of future cell-phones.

Resonance Raman Spectra for the In Situ Identification of Bacteria Strains and Their Inactivation Mechanism.

The resonance Raman spectra of bacterial carotenoids have been employed to identify bacterial strains and their intensity changes as a function of ultraviolet (UV) radiation dose have been used to differentiate between live and dead bacteria. In addition, the resonance-enhanced Raman spectra enabled us to detect bacteria in water at much lower concentrations (∼108 cells/mL) than normally detected spectroscopically. A handheld spectrometer capable of recording resonance Raman spectra in situ was designed, constructed, and was used to record the spectra. In addition to bacteria, the method presented in this paper may also be used to identify fungi, viruses, and plants, in situ, and detect infections within a very short period of time.

Thymine dissociation and dimer formation: A Raman and synchronous fluorescence spectroscopic study.

In this study, absorption, fluorescence, synchronous fluorescence, and Raman spectra of nonirradiated and ultraviolet (UV)-irradiated thymine solutions were recorded in order to detect thymine dimer formation. The thymine dimer formation, as a function of irradiation dose, was determined by Raman spectroscopy. In addition, the formation of a mutagenic (6-4) photoproduct was identified by its synchronous fluorescence spectrum. Our spectroscopic data suggest that the rate of conversion of thymine to thymine dimer decreases after 20 min of UV irradiation, owing to the formation of an equilibrium between the thymine dimers and monomers. However, the formation of the (6-4) photoproduct continued to increase with UV irradiation. In addition, the Raman spectra of nonirradiated and irradiated calf thymus DNA were recorded, and the formation of thymine dimers was detected. The spectroscopic data presented make it possible to determine the mechanism of thymine dimer formation, which is known to be responsible for the inhibition of DNA replication that causes bacteria inactivation.

A novel approach for remote detection of bacteria using simple charge-coupled device cameras and telescope.

We have designed, constructed, and utilized a charge-coupled device system, integrated with a small Newtonian telescope, capable of long distance recording of bacterial fluorescence and synchronous spectra for the detection of bacteria, their component molecules, and other species. This newly developed optical system utilizes commercial monochrome cameras that we have used to detect various bacterial strains, such as Escherichia coli, and determine their concentrations. In addition, using this system, we were able to differentiate between live and dead bacteria after treatment with ultraviolet light or antibiotics.

Ultrafast time-resolved structural changes of thin-film ferromagnetic metal heated with femtosecond optical pulses.

As a classic ferromagnetic material, nickel has been an important research candidate used to study dynamics and interactions of electron, spin, and lattice degrees of freedom. In this study, we specifically chose a thick, 150 nm ferromagnetic nickel (111) single crystal rather than 10-20 nm thin crystals that are typically used in ultrafast studies, and we revealed both the ultrafast heating within the skin depth and the heat transfer from the surface (skin) layer to the bulk of the crystal. The lattice deformation after femtosecond laser excitation was investigated by means of 8.04 keV subpicosecond x-ray pulses, generated from a table-top laser-plasma based source. The temperature evolution of the electron, spin, and lattice was determined using a three temperature model. In addition to coherent phonon oscillations, the blast force and sonic waves, induced by the hot electron temperature gradient, were also observed by monitoring the lattice contractions during the first couple of picoseconds after laser irradiation. This study further revealed the tens of picoseconds time required for heating the hundred nanometer bulk of the Ni (111) single crystals.

A tryptophan synchronous and normal fluorescence study on bacteria inactivation mechanism.

The UV photodissociation kinetics of tryptophan amino acid, Trp, attached to the membrane of bacteria, Escherichia coli and Bacillus subtilis, have been studied by means of normal and synchronous fluorescence. Our experimental data suggest that the fluorescence intensity of Trp increases during the first minute of irradiation with 250 nm to ∼ 280 nm, 7 mW/cm2 UV light, and subsequently decreases with continuous irradiation. During this short, less than a minute, period of time, 70% of the 107 cell per milliliter bacteria are inactivated. This increase in fluorescence intensity is not observed when tryptophan is in the free state, namely, not attached to a protein, but dissolved in water or saline solution. This increase in fluorescence is attributed to the additional fluorescence of tryptophan molecules formed by protein unfolding, the breakage of the bond that attaches Trp to the bacterial protein membrane, or possibly caused by the irradiation of 2 types of tryptophan residues that photolyze with different quantum yields.

Determination of live:dead bacteria as a function of antibiotic treatment.

Antibiotics are drugs that react against, kill, or inhibit the growth of bacteria. The method most often employed to evaluate the effectiveness of an antibiotic to kill bacteria requires at least 16 to 24 h for bacterial incubation. The requirement of long periods of time for the determination of the number of bacteria still alive after antibiotic treatment, may, in many cases, be detrimental to the patient's health. In addition, with increasing of bacterial antibiotic resistance, the need to utilize methods for distinguishing between live and dead bacteria within a short period of time after treatment with antibiotic agents, is becoming more crucial. To that effect, we have utilized a hand-held double monochromator to record in situ and within minutes the synchronous and normal fluorescence spectra of bacteria and other species. The fluorescence spectra of bacterial components such as tryptophan, tyrosine and DNA are clearly displayed. In addition, principal component analysis, PCA, makes it possible to display live and dead bacteria separately and determine the ratio of live:dead bacteria before and after treatment with antibiotics.

In situ detection of live-to-dead bacteria ratio after inactivation by means of synchronous fluorescence and PCA.

The determination of live and dead bacteria is of considerable significance for preventing health care-associated infection in hospitals, field clinics, and other areas. In this study, the viable (live) and nonviable (dead) bacteria in a sample were determined by means of their fluorescence spectra and principal component analysis (PCA). Data obtained in this study show that it is possible to identify bacteria strains and determine the live/dead ratio after UV light inactivation and antibiotic treatment, in situ, within minutes. In addition, synchronous fluorescence scans enable the identification of bacterial components such as tryptophan, tyrosine, and DNA. Compared with the time-consuming plating and culturing methods, this study renders a means for rapid detection and determination of live and dead bacteria.

Hand-held synchronous scan spectrometer for in situ and immediate detection of live/dead bacteria ratio.

The design, construction, and operation of a hand-held synchronously scanned, excitation-emission, double monochromator spectrometer is described. Data show that it is possible to record and display within minutes the fluorescence spectra and ratio of live/dead bacteria in situ. Excitation emission matrix contour plots display clearly bacteria fluorescence spectra before and after UV inactivation, respectively. The separation of the fluorescence band maxima of molecular components, such as tryptophan, tyrosine, and DNA, may be distinguished in the diffused fluorescence spectra of bacteria and mixtures.

Enhanced limonene production in cyanobacteria reveals photosynthesis limitations.

Terpenes are the major secondary metabolites produced by plants, and have diverse industrial applications as pharmaceuticals, fragrance, solvents, and biofuels. Cyanobacteria are equipped with efficient carbon fixation mechanism, and are ideal cell factories to produce various fuel and chemical products. Past efforts to produce terpenes in photosynthetic organisms have gained only limited success. Here we engineered the cyanobacterium Synechococcus elongatus PCC 7942 to efficiently produce limonene through modeling guided study. Computational modeling of limonene flux in response to photosynthetic output has revealed the downstream terpene synthase as a key metabolic flux-controlling node in the MEP (2-C-methyl-d-erythritol 4-phosphate) pathway-derived terpene biosynthesis. By enhancing the downstream limonene carbon sink, we achieved over 100-fold increase in limonene productivity, in contrast to the marginal increase achieved through stepwise metabolic engineering. The establishment of a strong limonene flux revealed potential synergy between photosynthate output and terpene biosynthesis, leading to enhanced carbon flux into the MEP pathway. Moreover, we show that enhanced limonene flux would lead to NADPH accumulation, and slow down photosynthesis electron flow. Fine-tuning ATP/NADPH toward terpene biosynthesis could be a key parameter to adapt photosynthesis to support biofuel/bioproduct production in cyanobacteria.

Synergistic reaction of silver nitrate, silver nanoparticles, and methylene blue against bacteria.

In this paper we describe the antibacterial effect of methylene blue, MB, and silver nitrate reacting alone and in combination against five bacterial strains including Serratia marcescens and Escherichia coli bacteria. The data presented suggest that when the two components are combined and react together against bacteria, the effects can be up to three orders of magnitude greater than that of the sum of the two components reacting alone against bacteria. Analysis of the experimental data provides proof that a synergistic mechanism is operative within a dose range when the two components react together, and additive when reacting alone against bacteria.

The low photo-inactivation rate of bacteria in human plasma II. Inhibition of methylene blue bleaching in plasma and effective bacterial destruction by the addition of dilute acetic acid to human plasma.

Methylene blue (MB) and other photo-sensitizer molecules have been recognized as effective means for the inactivation of bacteria and other pathogens owing to their ability to photo-generate reactive oxygen species (ROS) including singlet oxygen. These reactive species react with the membrane of the bacteria causing their destruction. However, the efficiency of MB to destroy bacteria in plasma is very low because the MB 660 nm absorption band, that is responsible for the ROS generation, is bleached. The bleaching of MB, in plasma, is caused by the attachment of a hydrogen atom to the central ring nitrogen of MB, which destroys the ring conjugation and forms Leuco-MB which does not absorb in the 600 nm region. In this paper we show that addition of dilute acetic acid, ∼10(-4) M, to human plasma, prevents H-atom attachment to MB, allowing MB to absorb at 660 nm, generates singlet oxygen and thus inactivates bacteria. The mechanism proposed, for preventing MB bleaching in plasma, is based on the oxidation of cysteine to cystine, by reaction with added dilute acetic acid, thus eliminating the availability of the thiol hydrogen atom which attaches to the MB nitrogen. It is expected that the addition of acetic acid to plasma will be effective in the sterilization of plasma and killing of bacteria in wounds and burns.

Subpicosecond and Sub-Angstrom Time and Space Studies by Means of Light, X-ray, and Electron Interaction with Matter.

This Perspective article considers an experimental system that consists of ultrafast optical, electron, and X-ray time-resolved components. These techniques are used simultaneously on the same sample to study, in real time, the events that occur immediately upon disturbance with an ultrafast optical pulse. Excited states and metastable species are generated on the surface, and the electrical and mechanical waves propagating through the sample are recorded with subpicosecond and sub-Angstrom resolution. The characteristic of each technique is briefly described as a means of introducing the experimental system that intergrates these techniques. The processes evolved after femtosecond excitation of a Au single crystal have been monitored by these techniques. The data presented show changes with a resolution of 0.3 ± 0.1 ps in optical thermoreflectance, 1.0 ± 0.2 ps in electron Bragg diffraction, and 0.6 ± 0.1 ps in X-ray diffraction intensity accompanying shift and broadening.

Rationale and mechanism for the low photoinactivation rate of bacteria in plasma.

The rate of bacterial photoinactivation in plasma by methylene blue (MB), especially for Gram-negative bacteria, has been reported to be lower, by about an order of magnitude, than the rate of inactivation in PBS and water solutions. This low inactivation rate we attribute to the bleaching of the 660-nm absorption band of MB in plasma that results in low yields of MB triplet states and consequently low singlet oxygen generation. We have recorded the change of the MB 660-nm-band optical density in plasma, albumin, and cysteine solutions, as a function of time, after 661-nm excitation. The transient triplet spectra were recorded and the singlet oxygen generated in these solutions was determined by the rate of decrease in the intensity of the 399-nm absorption band of 9, 10-anthracene dipropionic acid. We attribute the bleaching of MB, low singlet oxygen yield, and consequently the low inactivation rate of bacteria in plasma to the attachment of a hydrogen atom, from the S-H group of cysteine, to the central nitrogen atom of MB and formation of cysteine dimer.

Electron transfer mechanism in organometallic molecules studied by subpicosecond extended X-ray absorption fine structure spectroscopy.

The mechanism responsible for the redox reaction of [Co(III)(en)3]Ac3 to Co(II) complex has been determined to be intramolecular electron transfer. It was measured in real time by means of subpicosecond extended X-ray absorption fine structure spectra, EXAFS, and optical experiments and supported by density functional theory calculations. The proposed mechanism is based on histograms of bond length changes of the transient structures measured as a function of time, with subpicosecond time and sub-Angstrom resolution and femtosecond transient spectra and kinetics after excitation with a 267 nm femtosecond pulse. Even though four Fe and Co complexes were excited in the charge transfer band and the photoinduced redox reaction proceeds with similar high redox quantum yield, the dominant electron operating mechanism differs: intramolecular for amine metal complexes and intermolecular for oxalate metal complexes. The ligand orientation degree of freedom and counterion effect are proposed to provide tentative explanation for the electron transfer mechanism.

Time-resolved structural dynamics of thin metal films heated with femtosecond optical pulses.

We utilize 100 fs optical pulses to induce ultrafast disorder of 35- to 150-nm thick single Au(111) crystals and observe the subsequent structural evolution using 0.6-ps, 8.04-keV X-ray pulses. Monitoring the picosecond time-dependent modulation of the X-ray diffraction intensity, width, and shift, we have measured directly electron/phonon coupling, phonon/lattice interaction, and a histogram of the lattice disorder evolution, such as lattice breath due to a pressure wave propagating at sonic velocity, lattice melting, and recrystallization, including mosaic formation. Results of theoretical simulations agree and support the experimental data of the lattice/liquid phase transition process. These time-resolved X-ray diffraction data provide a detailed description of all the significant processes induced by ultrafast laser pulses impinging on thin metallic single crystals.