The relaxation dynamics of the excited electronic states of retinal in bacteriorhodopsin by two-pump-probe femtosecond studies.

We present the results of two-pump and probe femtosecond experiments designed to follow the relaxation dynamics of the lowest excited state (S(1)) populated by different modes. In the first mode, a direct (S(0) --> S(1)) radiative excitation of the ground state is used. In the second mode, an indirect excitation is used where the S(1) state is populated by the use of two femtosecond laser pulses with different colors and delay times between them. The first pulse excites the S(0) --> S(1) transition whereas the second pulse excites the S(1) --> S(n) transition. The nonradiative relaxation from the S(n) state populates the lowest excited state. Our results suggest that the S(1) state relaxes faster when populated nonradiatively from the S(n) state than when pumped directly by the S(0) --> S(1) excitation. Additionally, the S(n) --> S(1) nonradiative relaxation time is found to change by varying the delay time between the two pump pulses. The observed dependence of the lowest excited state population as well as its dependence on the delay between the two pump pulses are found to fit a kinetic model in which the S(n) state populates a different surface (called S'(1)) than the one being directly excited (S(1)). The possible involvement of the A(g) type states, the J intermediate, and the conical intersection leading to the S(0) or to the isomerization product (K intermediate) are discussed in the framework of the proposed model.

Cavity ringdown detection of losses in thin films in the telecommunication wavelength window.

The method of cavity ringdown spectroscopy (when a tunable pulsed optical parametric oscillator was used) was extended for the loss evaluation in thin films (2-20-microm thickness). The technique was applied in two key telecommunication wavelength ranges of 1260-1330 and 1480-1650 nm. The measurement sensitivity was determined to be 50 ppm (5 x 10(-5)). The results for polymer films are in close correlation with conventional spectrophotometric data and propagation loss for planar waveguides. Films of greater thickness and better optical quality are expected to provide an even higher loss resolution.

Catalysis of the retinal subpicosecond photoisomerization process in acid purple bacteriorhodopsin and some bacteriorhodopsin mutants by chloride ions.

The dynamics and the spectra of the excited state of the retinal in bacteriorhodopsin (bR) and its K-intermediate at pH 0 was compared with that of bR and halorhodopsin at pH 6.5. The quantum yield of photoisomerization in acid purple bR was estimated to be at least 0.5. The change of pH from 6.5 to 2 causes a shift of the absorption maximum from 568 to 600 nm (acid blue bR) and decreases the rate of photoisomerization. A further decrease in pH from 2 to 0 shifts the absorption maximum back to 575 nm when HCl is used (acid purple bR). We found that the rate of photoisomerization increases when the pH decreases from 2 to 0. The effect of chloride anions on the dynamics of the retinal photoisomerization of acid bR (pH 2 and 0) and some mutants (D85N, D212N, and R82Q) was also studied. The addition of 1 M HCl (to make acid purple bR, pH 0) or 1 M NaCl to acid blue bR (pH 2) was found to catalyze the rate of the retinal photoisomerization process. Similarly, the addition of 1 M NaCl to the solution of some bR mutants that have a reduced rate of retinal photoisomerization (D85N, D212N, and R82Q) was found to catalyze the rate of their retinal photoisomerization process up to the value observed in wild-type bR. These results are explained by proposing that the bound Cl- compensates for the loss of the negative charges of the COO- groups of Asp85 and/or Asp212 either by neutralization at low pH or by residue replacement in D85N and D212N mutants.

Replacement effects of neutral amino acid residues of different molecular volumes in the retinal binding cavity of bacteriorhodopsin on the dynamics of its primary process.

We have determined the rate and quantum yield of retinal photoisomerization, the spectra of the primary transients, and the energy stored in the K intermediate in the photocycle of some bacteriorhodopsin mutants (V49A, A53G, and W182F) in which residue replacements are found to change the Schiff base deprotonation kinetics (and thus the protein-retinal interaction). Because of their change in the local volume resulting from these individual replacements, these substitutions perturb the proton donor-acceptor relative orientation change and thus the Schiff base deprotonation kinetics. These replacements are thus expected to change the charge distribution around the retinal, which controls its photoisomerization dynamics. Subpicosecond transient spectroscopy as well as photoacoustic technique are used to determine the retinal photoisomerization rate, quantum yield, and the energy stored in the K-intermediate for these mutants. The results are compared with those obtained for wild-type bacteriorhodopsin and other mutants in which charged residues in the cavity are replaced by neutral ones. In some of the mutants the rate of photoisomerization is changed, but in none is the quantum yield or the energy stored in the K intermediate altered from that in the wild type. These results are discussed in terms of the shapes of the potential energy surfaces of the excited and ground states of retinal in the perpendicular configuration within the protein and the stabilization of the positive charge in the ground and the excited state of the electronic system of retinal.

Spot size reduction in coupling of laser to micromanipulator in laser microsurgery by fiberoptic link.

BACKGROUND AND OBJECTIVE: In coupling laser with micromanipulator through fiberoptics, the resulting diameter of the spot is limited by the laws of geometrical optics, because of the high numerical aperture (N.A.) of fiberoptic radiation. A new method for the reduction of spot size diameter is suggested. STUDY DESIGN/MATERIALS AND METHODS: The output of a 2 mW He-Ne laser was couped via fiberoptic link, the fiberoptic output light collected by a single lens collimator and directed to the input of the micromanipulator. RESULTS: The spot size can be considerably reduced by the introduction of an aperture which reduces the numerical aperture (N.A.) of the fiberoptic. The resulting reduction in total power has little effect on the power density. CONCLUSION: This approach to the collimator design permits reduction in spot size without any significant changes in power density, thus avoiding damage to the tissue and obtaining optimum performance from the micromanipulator.

The pH dependence of the subpicosecond retinal photoisomerization process in bacteriorhodopsin: evidence for parallel photocycles.

The pH dependence of the subpicosecond decay of the retinal photoexcited state in bacteriorhodopsin (bR) is determined in the pH range 6.8-11.3. A rapid change in the decay rate of the retinal photoexcited state is observed in the pH range 9-10, the same pH range in which a rapid change in the M412 formation kinetics was observed. This observation supports the previously proposed heterogeneity model in which parallel photocycles contribute to the observed pH dependence of the M412 formation kinetics in bR.

Effect of replacing the primary quinone by different species on the ultrafast photosynthetic electron transfer in bacterial reaction centres.

Using picosecond absorption spectroscopy it has been shown that in Rhodobacter sphaeroides reaction centres the substitution of the primary quinone acceptor (QA), ubiquinone-10, by other quinone species (with redox potentials higher or lower than that of ubiquinone-10) has essentially no modifying effect on the reaction centre protein. The molecular relaxation processes that accompany the localization and stabilization of a photo-excited electron on the intermediate acceptor, bacteriopheophytin (I), are not affected, although the subsequent transfer of the electron from I to QA is slowed down. Consequently, this leads to a lower quantum efficiency of high rate of direct I-----QA reaction is normally due to the specificity of the primary quinone species and its binding site in the reaction centre protein which provide optimum steric and chemical conditions for an effective interaction between I and QA.