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.

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.